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Granulocyte colony-stimulating factor exacerbates cardiac fibrosis after myocardial infarction in a rat model of permanent occlusion

Zhaokang Cheng, Lailiang Ou, Yi Liu, Xiaolei Liu, Fei Li, Bin Sun, Yongzhe Che, Deling Kong, Yaoting Yu, Gustav Steinhoff
DOI: http://dx.doi.org/10.1093/cvr/cvn202 425-434 First published online: 1 August 2008

Abstract

Aims Controversy exists regarding the effects of granulocyte colony-stimulating factor (G-CSF) on post-infarction remodelling, which is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate the impact of G-CSF administration on cardiac MMP/TIMP ratios and long-term remodelling in a rat model of acute myocardial infarction (MI).

Methods and results Sprague–Dawley rats underwent coronary ligation to produce MI. Rats surviving the MI for 3 h were randomized to receive G-CSF (50 µg/kg/day for 5 consecutive days, n = 16) or saline (n = 10). Sham-operated animals received no treatment (n = 10). G-CSF injection significantly increased circulating white blood cells, neutrophils, and monocytes. Western blotting revealed that the ratios of MMP-2/TIMP-1 and MMP-9/TIMP-1 were significantly decreased in the infarcted myocardium. At 3 months, echocardiographic and haemodynamic examinations showed that the G-CSF treatment induced left ventricular (LV) enlargement and dysfunction. Histological analysis revealed that the extent of myocardial fibrosis and infarct size were larger in the G-CSF group than in the Saline group. Furthermore, G-CSF treated animals showed a significantly lower post-MI survival during the study period.

Conclusion Decrease of cardiac MMP/TIMP ratios by G-CSF after infarction may be important as a mechanism in promotion of myocardial fibrosis, which further facilitates infarct expansion and LV dysfunction.

Keywords
  • G-CSF
  • Myocardial infarction
  • Fibrosis
  • Infarct expansion
  • Remodelling

1. Introduction

Myocardial infarction (MI), characterized by rapid death of cardiomyocytes and vascular structures, leads to ventricular remodelling and heart failure. The injection of bone marrow stem cells in animals with MI has been shown to restore the infarcted myocardium and improve cardiac performance.1 Our colleagues previously reported that the transplantation of bone marrow-derived mesenchymal stem cells overexpressing the prosurvival gene Akt reduces ventricular remodelling and improves cardiac function.2 However, the invasive nature of intramyocardial injection limits its potential application in the clinical setting.

Subcutaneous injection of granulocyte colony-stimulating factor (G-CSF), a non-invasive approach, can mobilize haematopoietic stem cells (HSCs) from bone marrow into the peripheral blood circulation.3 We previously reported that the G-CSF pre-treatment increases circulating endothelial progenitor cells (EPCs) and enhances repair of injured arteries in a balloon-injury rat model.4 However, both experimental studies58 and clinical trials914 showed conflicting results concerning effects of G-CSF in post-infarct left ventricular (LV) remodelling and function.

Recently, Sugano et al.15 demonstrated that G-CSF facilitated fibrosis in the infarcted myocardium, but the mechanism was still unclear. Matrix metalloproteinases (MMPs), the principal matrix-degrading proteinases, and their tissue inhibitors (TIMPs), are of great importance in cardiac remodelling.16 The possibility that G-CSF might affect post-MI remodelling by regulating the enzymatic systems of proteolysis and antiproteolysis has not been well investigated yet. Thus, the purpose of this study was to evaluate the effect of G-CSF on MMP/TIMP profiles and long-term ventricular remodelling in a rat model of MI.

2. Methods

2.1 Animals

Sprague–Dawley (SD) rats were purchased from the Laboratory Animal Center of The Academy of Military Medical Sciences (Beijing, China). The animal studies were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Tianjin, revised in June 2004), which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Protocol I: long-term effect of granulocyte colony-stimulating factor

2.2.1 Myocardial infarction and granulocyte colony-stimulating factor administration

MI was induced in female SD rats (n = 29) weighing 200–230 g by coronary ligation as previously described.2 After left thoracotomy and pericardiotomy, permanent occlusion of the left anterior descending (LAD) coronary artery was created with a 6–0 silk suture. Sham-operated animals underwent the same procedure but the coronary ligature was left untied (Sham group, n = 10). MI rats surviving the operation for 3 h (n = 26) were randomly assigned to two groups: (i) recombinant human G-CSF (50 µg/kg/day, Jiuyuan Gene Engineering Co. Ltd., Hangzhou, China) administered subcutaneously for 5 days (G-CSF group, n = 16), and (ii) saline-treated control (Saline group, n = 10).

2.2.2 Echocardiography

Echocardiographic studies were performed 1 and 3 months after LAD ligation by an experienced echocardiographer who was blinded to the group, using a Technos MPX DU8 system equipped with a 12.5–5.5 MHz broadband linear-array transducer (Esaote, Genoa, Italy).

2.2.3 Haemodynamics

Haemodynamic studies were performed 3 months after coronary ligation. A 2F high fidelity, microtip pressure catheter (model SPR-320, Millar Instruments, Houston, TX, USA) was placed in the right carotid artery and then advanced retrogradely into the LV. Haemodynamic parameters were recorded using the Biopac physiological data acquisition system (MP150, Biopac Systems, Inc., Goleta, CA, USA).

2.2.4 Histopathology

After haemodynamic measurement, the animals were sacrificed and their hearts were excised for histological analysis. The hearts were immersion-fixed in Carnoy's fluid and embedded in paraffin. The tissues were cut into 5 µm-thick sections. Standardized transversal median ventricular sections stained with haematoxylin-eosin and picrosirius red were studied by a single investigator who was unaware of the nature of the experimental groups. The infarct size was determined as the percentage of infarcted epicardium and endocardium of the LV circumference. The minimal thickness of LV free wall (LVFWTmin) was measured to evaluate the risk of ventricular rupture. Percent fibrosis was expressed as the ratio of fibrotic area to total LV area. Collagen volume fraction (CVF) in the infarct scar was measured by using polarization microscopy in five fields for each LV section. All morphological analysis was performed by using Image-Pro Plus software (Version 4.5, Media Cybernetics, Silver Spring, MD, USA).

2.2.5 Immunohistochemistry

Representative tissue sections (5 µm) from each animal were deparaffinized, rehydrated, and incubated with Isolectin IB4 Alexa Fluor 488 dye conjugate (Invitrogen, Carlsbad, CA, USA), or a rabbit polyclonal antibody to the endothelial cell marker von Willebrand factor (vWF; DakoCytomation, Glostrup, Denmark) followed by FITC-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA, USA). Quantification of vessel density (vessels/mm2) was performed by counting capillaries and arterioles in 15 randomly selected fields concerning the remote and infarct regions using a fluorescent microscope (Olympus BX-41, Tokyo, Japan).

2.3 Protocol II: short-term effect of granulocyte colony-stimulating factor

In this protocol, another set of animals in the Sham (n = 4), Saline (n = 5), and G-CSF groups (n = 5) were prepared.

2.3.1 Blood sampling

Three hours after the last G-CSF injection, blood samples (2 mL each) were collected from abdominal aorta for peripheral blood cell counts.

2.3.2 Histological examination

Five days after MI, 5 µm paraffin sections were prepared. To examine the recruitment of stem cells, heart sections were incubated with the stem/progenitor cell marker, polyclonal rabbit anti-c-kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA, USA). To examine the infiltration of leukocytes, sections were stained with haematoxylin-eosin. Ten fields concerning the remote and infarct regions were randomly chosen from each section. The number of c-kit+ cells or leukocytes was expressed as cells/high power field (400×).

2.3.3 Western blotting

Immediately after blood sampling, the LV infarct region was separated and immediately frozen in liquid nitrogen. Fifty milligrams of frozen tissues from each heart were homogenized in lysis buffer and centrifuged for 10 min at 14 000 rpm and 4°C. Denatured proteins (40 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a PVDF membrane. After blocking with 5% skim milk in tris-buffered saline containing tween-20, the membranes were incubated with rabbit anti-MMP-2, rabbit anti-MMP-9, rabbit anti-TIMP-1, mouse anti-TIMP-2, rabbit anti-transforming growth factor β (TGFβ), or rabbit anti-actin (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Zymed, San Francisco, CA, USA) was used as the secondary antibody. Hybridizing bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). The signals were quantified by densitometry.

2.4 Protocol III: effect of granulocyte colony-stimulating factor on normal hearts

2.4.1 Animal groups, functional and histological examination

To examine the effects of G-CSF on normal hearts, sham-operated rats were treated with 50 µg/kg/day G-CSF for 5 days (Sham + G-CSF group, n = 9) or received no additional treatment (Sham group, n = 9). After 30 days, heart function, CVF, and cardiomyocyte hypertrophy were evaluated by echocardiography, picrosirius red staining, and wheat germ agglutinin staining, respectively (for detailed information, see Supplementary material online).

2.5 Statistical analysis

Data are expressed as mean ± SEM. Mortality was analysed by means of the Kaplan–Meier method, and the between-group difference in survival was tested by the log-rank (Mantel-Cox) test. Comparison between two groups was performed with the unpaired Student t-test. Multiple comparisons were performed by one-way ANOVA. Bonferroni (equal variances) and Dunnett's T3 (unequal variances) post hoc tests were used to specify pair-wise differences. A value of P < 0.05 was considered statistically significant.

3. Results

3.1 Peripheral blood cell counts

Following daily subcutaneous G-CSF for 5 days, white blood cells, neutrophils, and monocytes were significantly increased when compared to the Saline group (Table 1). There was no effect of G-CSF on the number of red blood cells and platelets, or on the level of haemoglobin. Additionally, the number of lymphocytes was decreased by G-CSF treatment (Table 1).

View this table:
Table 1

Peripheral blood cell counts examined at 3 h after the last granulocyte colony-stimulating factor injection

ParametersShamSalineG-CSF
White blood cells, ×109 L−13.87 ± 0.374.66 ± 0.327.49 ± 0.75*
  Neutrophils, ×109 L−11.63 ± 0.122.78 ± 0.13*6.11 ± 0.70*
 % in WBC41.87 ± 4.0259.50 ± 1.44*80.74 ± 2.30*
  Monocytes, ×107 L−12.51 ± 0.126.69 ± 1.4426.00 ± 2.69*
 % in WBC0.67 ± 0.091.38 ± 0.233.61 ± 0.40*
  Lymphocytes, ×109 L−12.23 ± 0.321.82 ± 0.181.11 ± 0.13*
 % in WBC57.47 ± 4.0839.02 ± 1.30*15.57 ± 2.21*
Red blood cells, ×1012 L−16.81 ± 0.736.42 ± 0.386.42 ± 0.15
Platelets, ×109 L−11100 ± 98878 ± 49822 ± 45*
Haemoglobin, g/L140 ± 8140 ± 7137 ± 3
  • WBC, white blood cells.

  • Values are mean ± SEM.

  • *P < 0.05 vs. Sham.

  • P < 0.05 vs. Saline.

3.2 Infiltration of c-kit+ cells and leukocytes in the damaged heart

We examined c-kit+ cells and leukocytes in the damaged heart 5 days post MI. Ligation of the coronary artery induced significant infiltration of c-kit+ cells and leukocytes in the infarcted myocardium. The number of c-kit+ cells or leukocytes in the infarct region was not significantly changed by G-CSF treatment (Figure 1). In the remote non-infarcted region, there was no significant difference in the number of c-kit+ cells or leukocytes among the three groups (data not shown).

Figure 1

Infiltration of c-kit+ cells and leukocytes in the infarcted myocardium. (A) Representative heart sections of the three groups of animals after c-kit immunostaining (top) or haematoxylin-eosin staining (bottom). Green fluorescence represents the transmembrane c-kit antigen. Arrows indicate infiltrating leukocytes. Scale bar = 20 µm. (B and C) The number of c-kit+ cells (B) or leukocytes (C) in the infarcted myocardium did not differ significantly between saline- and G-CSF-treated animals. *P < 0.05 vs. Sham.

3.3 Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and transforming growth factor-β in the infarcted myocardium

MMP-2 is recognized by the bands at 72 kDa (pro-form), 68 kDa, and 54 kDa (active-form),17 and MMP-9 at the 83 kDa (intermediate form), 67 kDa, and 64 kDa (active-form) (Figure 2A).18 A band representing the pro-form of MMP-9 (92 kDa) was not observed in this study. At 5 days after MI, total MMP-9 in the saline-treated animals was significantly increased by >150% and total MMP-2 showed a trend to increase when compared to the Sham group (Figure 2B, top). The upregulation of MMP-2 and MMP-9 were inhibited by G-CSF treatment. In contrast, the expression of TIMP-1 (28 kDa) was significantly higher in G-CSF-treated animals than in saline-treated animals. As a consequence, the ratios of MMP-2/TIMP-1 and MMP-9/TIMP-1 were significantly reduced by >60% in the G-CSF-treated animals (Figure 2B, bottom). No statistical difference in the level of TIMP-2 (21 kDa) was observed between the G-CSF and Saline groups. However, MMP-2/TIMP-2 and MMP-9/TIMP-2 ratios also showed a trend to decrease by G-CSF injection. The expression of TGFβ in the Saline group tended to be higher 5 days after MI when compared with that of sham-operated animals, and was not affected by G-CSF treatment (Figure 2C and D).

Figure 2

Expression of MMPs, TIMPs, and TGFβ in the infarcted myocardium. (A) Representative western blots for MMP-2, MMP-9, TIMP-1, TIMP-2, and β-actin. (B) Top panel: Relative abundance of MMP-2, MMP-9, TIMP-1, and TIMP-2 normalized to β-actin content on each blot. Bottom panel: Ratios of MMP-2/TIMP-1, MMP-2/TIMP-2, MMP-9/TIMP-1, and MMP-9/TIMP-2. (C) Representative western blots for TGFβ and β-actin. (D) Relative abundance of TGFβ normalized to β-actin content. Values are mean ± SEM. *P < 0.05 vs. Sham; P < 0.05 vs. Saline.

3.4 Echocardiography

We evaluated LV size, function, and wall thickness by echocardiography 1 and 3 months after the operation (Table 2). At both time-points, G-CSF-treated rats showed a marked increase in LV end-diastolic diameter (LVDd) and LV end-systolic diameters (LVDs) when compared to the Saline group. Consistently, LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) in the G-CSF group were larger than those in the Saline group 3 months after infarction. Although stroke volume (SV) showed a trend to increase by G-CSF treatment at 3 months, ejection fraction (EF) and fractional shortening (FS) were significantly decreased compared with saline-treated animals. Permanent ligation of the LAD significantly reduced the anterior wall thickness (AWT) and the interventricular septum thickness (IVST) but not the posterior wall thickness (PWT) in both saline-treated and G-CSF-treated animals 1 month after MI. Compared to 1 month, AWT, PWT were significantly increased and IVST showed a trend to increase in the Saline group at 3 months, whereas they remained statistically unchanged in the G-CSF group.

View this table:
Table 2

Echocardiographic data 1 and 3 months after infarction

Parameters1 month3 monthsP-value
AWT (mm)
 Sham1.5 ± 0.11.7 ± 0.2NS
 Saline1.0 ± 0.1*1.5 ± 0.2<0.03
 G-CSF0.9 ± 0.1*1.0 ± 0.1*NS
IVST (mm)
 Sham1.6 ± 0.11.7 ± 0.2NS
 Saline1.0 ± 0.1*1.3 ± 0.20.16
 G-CSF0.8 ± 0.1*0.9 ± 0.1*NS
PWT (mm)
 Sham1.7 ± 0.11.8 ± 0.1NS
 Saline1.6 ± 0.12.0 ± 0.05*<0.01
 G-CSF1.7 ± 0.11.8 ± 0.1NS
LVDd (mm)
 Sham6.9 ± 0.36.4 ± 0.2NS
 Saline7.6 ± 0.47.1 ± 0.5NS
 G-CSF11.5 ± 1.1*10.6 ± 0.5*NS
LVDs (mm)
 Sham3.7 ± 0.43.9 ± 0.4NS
 Saline5.2 ± 0.64.7 ± 0.8NS
 G-CSF9.1 ± 1.3*8.9 ± 0.4*NS
LVEDV (mm3)
 Sham249 ± 26212 ± 17NS
 Saline321 ± 37279 ± 45NS
 G-CSF800 ± 158*650 ± 62*NS
LVESV (mm3)
 Sham68 ± 1671 ± 13NS
 Saline148 ± 31134 ± 48NS
 G-CSF517 ± 134446 ± 46*NS
SV (mm3)
 Sham182 ± 19141 ± 14NS
 Saline174 ± 9145 ± 15NS
 G-CSF283 ± 40204 ± 17*NS
EF (%)
 Sham74 ± 567 ± 5NS
 Saline59 ± 662 ± 8NS
 G-CSF41 ± 9*32 ± 1*NS
FS (%)
 Sham47 ± 540 ± 5NS
 Saline35 ± 537 ± 6NS
 G-CSF23 ± 6*16 ± 0.4*NS
  • AWT, anterior wall thickness at end-diastole; IVST, interventricular septal thickness at end-diastole; PWT, posterior wall thickness at end-diastole; LVDd, LV end-diastolic diameter; LVDs, LV end-systolic diameter; LVEDV, LV end-diastolic volume; LVESV, LV end-systolic volume; SV, stroke volume; EF, ejection fraction; FS, fractional shortening; NS, not significant.

  • Values are mean ± SEM.

  • *P < 0.05 vs. Sham.

  • P < 0.05 vs. Saline.

3.5 Haemodynamics

We examined haemodynamic parameters 3 months after MI. LV dP/dtmax and dP/dtmin were significantly lower in the G-CSF group than in the Saline group (Figure 3A and B). Furthermore, G-CSF treatment tended to decrease the LV end-systolic pressure (LVESP) and increase the end-diastolic pressure (LVEDP) when compared with the Saline group (Figure 3C and D).

Figure 3

Haemodynamic parameters at 3 months after infarction. (A) dP/dtmax, maximal rate of pressure increase; (B) dP/dtmin, maximal rate of pressure decrease; (C) LVESP, LV end-systolic pressure; (D) LVEDP, LV end-diastolic pressure. Values are mean ± SEM. *P < 0.05 vs. Sham; P < 0.05 vs. Saline.

3.6 Histological analysis

Infarct size was markedly larger in the G-CSF treatment group than in the Saline group (Figure 4A and E). Compared to sham-operated animals, saline- and G-CSF-treated rats bearing MI showed a significantly decreased LVFWTmin 3 months after ligation. There was also a trend (although non-significant) toward smaller LVFWTmin in the G-CSF group than in the Saline group (Figure 4B and E). The percentage of fibrosis area in total LV area was significantly increased by G-CSF administration (Figure 4C and E). CVF in the infarct region also showed a trend to increase in the G-CSF group (Figure 4D and E).

Figure 4

Histological evaluation of left ventricular remodelling 3 months after infarction. (A) infarct size; (B) LVFWTmin, minimal thickness of LV free wall; (C) percent fibrosis; (D) collagen volume fraction in the infarct scar. Values are mean ± SEM. *P < 0.05 vs. Sham; P < 0.05 vs. Saline. (E) Representative sections stained with haematoxylin-eosin and picrosirius red observed under normal light (top and middle, scale bars = 2 mm) and polarized light (bottom, scale bar=200 µm).

3.7 Vessel density

Three months after surgery, vessel density was significantly reduced by coronary ligation in all infarcted animals. Vessel density in the infarct region was not significantly different between the Saline and G-CSF groups. However, vessel density in the remote region tended to be reduced by G-CSF treatment (Figure 5).

Figure 5

Assessment of vessel density 3 months after infarction. (A and B) Quantitative analysis of vessel density in the remote area (A) and infarct region (B). Values are mean ± SEM. *P < 0.05 vs. Sham. (C) Representative images of vessels in the remote area (top) and infarct region (bottom). Scale bar indicates 50 µm.

3.8 Mortality

Coronary artery ligation was performed in 29 rats. The peri-operative mortality rate, within 3 h after the operation, was 10.3% (three rats). Finally, 26 rats surviving the MI were randomly allocated to receive saline (10 rats) or G-CSF (16 rats). Three months after MI, G-CSF-treated rats showed a significant decrease in the survival rate compared to saline-treated animals (31.3 vs. 90.0%, P = 0.007, Figure 6).

Figure 6

Cumulative survival of MI rats treated with saline or G-CSF during the observation period of 3 months. Lifespan was estimated by the Kaplan–Meier method. Survival rate of G-CSF-treated rats was significantly lower than that of saline-treated rats (P < 0.01).

3.9 Effect of granulocyte colony-stimulating factor on normal hearts

Echocardiographic parameters, including LVDd, LVDs, EF, and FS, were similar in the Sham and Sham+G-CSF groups (Supplementary material online, Table S1). CVF in the sham-operated hearts was not significantly changed by G-CSF treatment (Supplementary material online, Figure S1). Cardiomyocyte hypertrophy was not identified in Sham + G-CSF group when compared with sham-operated animals (Supplementary material online, Figure S2).

4. Discussion

4.1 Main findings

G-CSF treatment immediately after acute MI increased circulating white blood cells (neutrophils and monocytes) and decreased MMP/TIMP stoichiometric ratios in the infarcted myocardium. In the long term, the administration of G-CSF exacerbated cardiac fibrosis, induced infarct expansion and ventricular dysfunction.

4.2 Effects of granulocyte colony-stimulating factor on myocardial infarction and proposed mechanisms

Stem cell transplantation has been proposed as a promising strategy for cardiac repair following MI.1 However, direct transplantation of stem cells to infarcted myocardium required a surgical intervention that was accompanied by high mortality and low success rate.5 Several haematopoietic cytokines, such as G-CSF, could mobilize HSCs and EPCs from bone marrow into the peripheral blood circulation3,4 by decreasing bone marrow concentration of SDF-1, a chemokine essential for the homing and retention of stem cells in the bone marrow.19,20 Thus, cytokine therapy was an attractive non-invasive strategy that might have great potential to repair the damaged myocardium. Subcutaneous injection of G-CSF and stem cell factor (SCF), demonstrated for the first time by Orlic et al.,5 regenerated the lost myocardium and increased EF by mobilization of primitive bone marrow cells. G-CSF mobilization of stem cells from bone marrow to infarcted myocardium was further confirmed by several groups using chimeric mice.2123 Mobilized stem cells were reported to improve LV function and regenerate infarcted myocardium by inducing myogenesis5,22,23 and vasculogenesis.5,2325 However, this concept has recently been challenged by studies in which mobilized bone marrow stem cells do not transdifferentiate to blood vessels or cardiomyocytes.26,27 Recently, the Komuro group6,25,28 showed that G-CSF benefits ischaemic myocardium by inhibiting apoptotic death of cardiomyocytes and endothelial cells, rather than stem cell mobilization. Another mechanism proposed by the Komuro group and others was that G-CSF increased vascular density in the ischaemic region6,25,28 by increasing neutrophils and their release of VEGF.29 Consistently, we observed more (but not significant) vessels in the infarct site after G-CSF administration. However, in the remote region, the vessel density tended to be lower. It does not appear to be due to vessel rarefaction, but rather to cardiomyocyte hypertrophy, as suggested by Kuhlmann et al.27

Although the beneficial effects of G-CSF on MI were observed in many studies,5,6,2328 controversy still exists. Deten et al.7 demonstrated that the administration of G-CSF and SCF failed to improve LV function and regenerate myocardium in a mouse MI model. Similar results were observed in rats,8 porcine,30 and baboons.31 Another closely related to cytokine, granulocyte–macrophage colony-stimulating factor (GM-CSF), showed no effect on infarct size and ventricular function in infarcted pigs.32 The GM-CSF induction by romurtide after MI deteriorated rat LV function and facilitated infarct expansion.33 In the present study, subcutaneous injection of G-CSF (50 µg/kg/day) started at 3 h post-MI for 5 days resulted in LV enlargement and dysfunction in a rat model of permanent ligation. The discrepancies between these studies may be explained by different dosage, timing, and duration of G-CSF therapy, administered alone or with other cytokines, different model (permanent ligation or reperfusion, with or without splenectomy) that were used. Furthermore, previous reports have suggested that animals of different species34 or at different ages35 may have different responses to G-CSF.

On the basis of early experimental studies, several clinical trials were performed and produced controversial results. The FIRSTLINE-AMI trial11 and a non-randomized study36 showed that G-CSF administration after successful recanalization of the infarct-related artery improved LV function and prevented remodelling. Another three randomized, double-blind, placebo-controlled trials, including STEMMI,12 G-CSF-STEMI,14 and REVIVAL-2,13 demonstrated that the G-CSF treatment after PCI was safe but had no influence on LV function. However, negative effects of G-CSF treatment following MI, such as high rate of in-stent restenosis,9,37 re-infarction, and death,10,37 were documented in other trials. Similarly, Zbinden et al.38 reported that two of seven patients treated with GM-CSF before PCI suffered an acute coronary syndrome during the treatment period. Concerning the adverse effects of G-CSF/GM-CSF on post-MI remodelling, several possible mechanisms were proposed. First, mobilized progenitor cells might participate in pathological vascular remodelling by differentiation into smooth muscle cells within the stented segment.9,37 Second, G-CSF/GM-CSF induced an exaggerated inflammatory response, based on peripheral monocytosis,33,38 infiltration of monocytes and macrophages into injured tissue,33,39 and upregulation of C-reactive protein (an index of systemic inflammation).10,14,36,40 It was reported that monocytosis might contribute to decreased myocardial perfusion,38 whereas C-reactive protein might provoke vascular inflammation.10 Thus, G-CSF injection following MI will induce vascular inflammation and promote myocardial damage. Furthermore, elevation of the white blood cell count during acute MI has been associated with an increased death rate.41 Thus, in the present study, the significantly increased circulating white blood cells might have contributed to the higher mortality in the G-CSF group. To study the effect of monocytosis on myocardial inflammation, we examined the infiltration of c-kit+ cells and leukocytes in the infarcted myocardium. Interestingly, the number of infiltrating c-kit+ cells or leukocytes in the myocardium was not changed by G-CSF irrespective of the effect on peripheral monocytosis. Therefore, other factors as arrhythmias or enhanced ischaemia by decreased microcirculatory perfusion in combination with cardiomyocyte hypertrophy may influence the increase in mortality by G-CSF. Our result is consistent with previous reports,15,21,24 which demonstrated that G-CSF had no effect on the infiltration of inflammatory cells. It was reported that inhibition of MMP-242 and MMP-943 suppressed leukocyte infiltration into the infarcted myocardium. Thus, our failure to see an increase in infiltrating leukocytes might be explained by G-CSF-mediated downregulation of MMPs. Third, G-CSF/GM-CSF treatment might influence fibrosis after infarction, but the precise effects were conflicting, with both increases15,26 and decreases28,33,44 being reported. Our results revealed that G-CSF exacerbated fibrosis and aggravated LV remodelling, whereas Sugano et al.15 showed that G-CSF promoted reparative fibrosis and attenuated early ventricular expansion by using a lower dosage (20 µg/kg/day) and an earlier detection time point (14 day post-MI). Cardiac fibrosis is the result of both exaggerated collagen synthesis and insufficient collagen degradation. TGFβ is a fibrogenic cytokine associated with collagen accumulation, whereas the balance between MMPs and TIMPs controls collagen degradation. Deten et al.45 demonstrated that TGFβ, MMP-2, MMP-9, and TIMP-2 were upregulated after MI. We further examined whether G-CSF influences the expression of these molecules. In agreement with Maekawa et al.,33 our results showed that the expression of TGFβ was not affected by G-CSF treatment 5 days after MI. However, the levels of MMP-2 and MMP-9 were decreased, and the expression of TIMP-1 was increased by G-CSF treatment. Downregulation of MMPs and upregulation of TIMPs would favour decreased collagen degradation and increased collagen accumulation.16 Thus, we conclude that G-CSF after MI enhances cardiac fibrosis by decreasing the MMP/TIMP ratios. It is known that TIMPs are produced primarily by fibroblasts, whereas MMP-2 and MMP-9 are produced by neutrophils and macrophages after MI.16 Thus, it is possible that G-CSF modulates the secretion of MMPs and TIMPs directly through its G-CSF receptor that is naturally expressed on cardiac fibroblasts,6 neutrophils, and monocytes.15

4.3 Study limitations

First, we used a model of permanent ligation in this study, which may not reflect the fact that most patients with acute MI undergo revascularization. However, adverse or no effects were also observed by others who administered the drug after reperfusion.1214,32,33,37 Second, G-CSF (50 µg/kg/day) was injected 3 h after MI and every 24 h thereafter for 5 days. During this phase, the infarcted myocardium is most vulnerable to various stimuli. Thus, it cannot be excluded that G-CSF at a different dose or timing may have a beneficial effect on post-MI remodelling. Third, we demonstrated that MMP/TIMP expression in the infarcted myocardium was changed by G-CSF, but the precise mechanism of this effect is still not clear. Further elucidation of the mechanisms is needed, including G-CSF-mediated stimulation of neutrophils/macrophages/fibroblasts, secretion of other inflammatory cytokines, and activation of cell-surface proteins and associated signal pathways, etc.

Conclusions

Administration of G-CSF in a rat model of permanent occlusion enhanced myocardial fibrosis, resulted in infarct expansion and aggravated LV remodelling. These adverse effects might be attributed to a decrease in MMP/TIMP ratios in the infarcted myocardium. Considering the potential application of G-CSF in treatment of MI, the profibrotic property of this cytokine should be considered in future trials.

Funding

National Science Foundation of China (No. 30570471); National Outstanding Youth Found (No. 30725030); Natural Science Foundation of Tianjin (No. 05YFJZJC01601); New Century Excellent Talent (NCET-04-0222); and China-Germany Project Based Personnel Exchange Program (PPP).

Acknowledgements

The authors thank Xin Zhou, Wei Pang, Shan Zeng (Institute of Cardiovascular Disease, Medical College of Armed Police Forces, Tianjin, China) and Boli Chen (Nankai University, China) for excellent technical assistance.

Conflict of interest: none declared.

References

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