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Cardiovascular Research 2006 70(1):61-69; doi:10.1016/j.cardiores.2005.12.013
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Copyright © 2006, European Society of Cardiology

Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice

Toru Yoshioka, Masafumi Takahashi*, Yuji Shiba, Chihiro Suzuki, Hajime Morimoto, Atsushi Izawa, Hirohiko Ise and Uichi Ikeda

Division of Cardiovascular Science, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan

* Corresponding author. Tel./fax: +81 263 37 3352/+81 263 37 2573. Email address: masafumi{at}sch.md.shinshu-u.ac.jp

Received 27 June 2005; revised 12 December 2005; accepted 14 December 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective Neointimal formation following percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since reendothelialization is one of the determinant factors for the development of neointimal formation, we examined the effects of granulocyte colony-stimulating factor (G-CSF) on reendothelialization and neointimal formation after vascular injury in mice.

Methods and results Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. G-CSF pretreatment significantly accelerated reendothelialization and decreased neointimal formation following vascular injury; however, this inhibitory effect of G-CSF was diminished when G-CSF was started following the injury. Flow cytometry analysis revealed that G-CSF treatment increased the number of endothelial progenitor cells (EPCs: CD34+/Flk-1+) in the peripheral circulation. Vascular injury was also produced in 2 types of mice whose bone marrow was replaced with that of enhanced green fluorescent protein- and Tie2/LacZ-transgenic mice. In the reendothelialized artery of these mice, few bone marrow-derived EPCs were detected. Furthermore, G-CSF treatment reduced the serum level of interleukin (IL)-6.

Conclusion G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI.

KEYWORDS Atherosclerosis; Cytokines; Restenosis; Stem cells

This article is referred to in the Editorial by J. Sainz and M. Sata (pages 3–5) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The vascular endothelium forms a biological interface between circulating blood components and various tissues in the body. This monolayer of endothelial cells monitors the stimuli generated systemically and locally and alters its functional state. This adaptive mechanism contributes to normal homeostasis; however, nonadaptive changes in the endothelial structure and function, provoked by pathophysiological stimuli, may induce ‘endothelial dysfunction’ that plays an important role in the initiation and progression of cardiovascular diseases [1,2]. In particular, the loss of endothelial cells by vascular injury leads to migration and proliferation of vascular smooth muscle cells, resulting in neointimal formation. The resultant neointimal formation is the pathological basis of restenosis that occurs after revascularization procedures such as angioplasty, stenting, and bypass grafting [3,4].

Granulocyte-colony stimulating factor (G-CSF) stimulates a marked increase in the migration of endothelial progenitor cells (EPCs) from the bone marrow into the peripheral blood circulation; this process is termed ‘mobilization’ [5,6]. EPCs have been shown to be recruited and incorporated into the site of neovascularization in ischemic tissues [7]. Further, they are known to have a therapeutic potential in hind limb ischemia [8–10] or cardiovascular diseases such as myocardial ischemia [11–15] and transplant arteriosclerosis [16]. Hence, G-CSF-mediated mobilization of EPCs could be a therapeutic strategy that promotes early reendothelialization after vascular injury and inhibits neointimal formation. At present, however, the effect of G-CSF treatment on neointimal formation after vascular injury remains controversial. Kang et al. [17] demonstrated that G-CSF promoted angiogenesis and improved cardiac function when administered in patients with coronary artery diseases (CAD); however, increased restenosis was observed as a serious adverse effect after percutaneous coronary intervention (PCI). In contrast, Kong et al. [18] reported that the G-CSF-induced mobilization of EPCs enhanced reendothelialization of the injured artery and inhibited neointimal formation in a rat balloon injury model. However, their study has two limitations: (1) the role of EPCs remains unclear because no bone marrow-transplanted animals were used and (2) the clinical feasibility of this approach needs to be evaluated without splenectomy because their observations were based on a splenectomized rat model. The purpose of the present study was to examine whether G-CSF treatment promotes reendothelialization and inhibits neointimal formation after vascular injury in intact mice, and to elucidate the role of bone marrow-derived EPCs in reendothelialization and neointimal formation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animals
Male C57BL/6 mice and FVB mice aged 8 or 12 weeks were purchased from Japan SLC Inc. (Hamamatsu, Japan) and CLEA Japan Inc. (Tokyo, Japan), respectively. C57BL/6 transgenic mice that ubiquitously express enhanced green fluorescent protein (GFP-Tg mice) were a generous gift from Dr. Masaru Okabe (Osaka University, Osaka, Japan), and FVB transgenic mice that express β-galactosidase under the control of the Tie2 promoter (Tie2/LacZ mice) were purchased from Jackson Laboratory (Bar Harbor, ME). All experiments in this study were performed in accordance with the Shinshu University Guide for Laboratory Animals which conforms to NIH Guidelines.

2.2 Experimental protocols
A total of 64 mice (age: 12 weeks old) were divided into the following three groups: (1) the first group was administered saline (vehicle group, n=20) for 10 days after vascular injury; (2) the second group was administered recombinant human G-CSF (100 µg/kg/day; Chugai Pharmaceutical, Co., Japan) for 10 days after vascular injury (G-CSF-posttreated group, n=22); and (3) the third group was administered G-CSF (100 µg/kg/day) for 4 consecutive days prior to vascular injury and for 6 days after the injury (G-CSF-pretreated group, n=22). Both saline and G-CSF were administered subcutaneously. In the vehicle or G-CSF-posttreated group, the first injection of saline or G-CSF was administered at 2 h after vascular injury. G-CSF was well tolerated by the mice and no abnormal behaviour was observed.

2.3 Bone marrow transplantation
Whole bone marrow cells were harvested by flushing femurs with phosphate-buffered saline (PBS). Red blood cells were lysed with ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2) at 4 °C for 20 min. The cells were washed three times with PBS and resuspended in 0.5 mL PBS. Recipient mice (age: 8 weeks old) were lethally irradiated with a total dose of 9 Gy (MBR-155R2; Hitachi, Japan) and were injected with bone marrow cells through the tail vein. After transplantation by this protocol, reconstitution of the bone marrow was verified by using GFP-Tg mice as donors. Flow cytometry analysis revealed that peripheral blood cells comprised more than 90% GFP-positive cells 8 weeks after bone marrow transplantation. We used C57BL/6 mice as recipients when the donors were GFP-Tg mice, and FVB mice were used as recipients when the donors were Tie2/LacZ mice.

2.4 Wire-mediated vascular injury
Wire-mediated vascular injury of the right femoral artery was produced by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15; Cook, Bloomington, IN), as previously described by Sata et al. [19]. We confirmed that this procedure induces similar neointimal formation in 8- to 16-week-old C57BL/6 and FVB mice.

2.5 Flow cytometry analysis
Blood samples were collected from the mice after G-CSF (100 µg/kg/day) or vehicle was administered for 4 consecutive days prior to vascular injury and 2 days after the injury. Circulating cells were identified using a nucleated cell fraction. The nucleated cells were double labelled with FITC-conjugated anti-CD34 monoclonal antibody (clone RAM34; BD Biosciences, San Jose, CA) and PE-conjugated anti-Flk-1 antibody (VEGFR2/KDR, clone Avas 12{alpha}1; BD Biosciences). The cells were examined by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed using CellQuest software ver. 3.3 (Becton Dickinson).

2.6 Histology
Three weeks after the vascular injury, mice were euthanized after irrigation with saline and blood was completely washed out. The femoral arteries were excised from each mouse, embedded in OCT compound (Tissue-Tek; Miles Laboratories, IN), and frozen in liquid nitrogen. Neointimal formation in the femoral arteries was evaluated at five locations separated by a distance of 100 µm, with the most distal site located at the point where the wire-inserted branch first appeared; these sites were stained with hematoxylin and eosin (HE). To quantify the neointimal lesions, each image was digitized and analyzed under a microscope (BX-51; Olympus, Tokyo, Japan) using NIH Image software ver. 1.63. The average value of five locations in each artery was determined.

2.7 Immunohistochemical analysis
Arterial sections, 10 µm in thickness, were incubated with primary antibodies against mouse CD31 (Becton Dickinson), GFP (MBL, Nagoya, Japan), and β-galactosidase (Rockland, Gilbertsville, PA), followed by incubation with biotin-conjugated secondary antibodies. The sections were washed and treated with avidin-peroxidase (ABC Elite kit; Vector Laboratories, Burlingame, CA). The reaction was developed by using a DAB substrate kit (Vector Laboratories). The sections were then counterstained with hematoxylin, mounted in glycerol, and examined under a light microscope. No signals were detected when irrelevant IgG was used instead of the primary antibody as a negative control. Endothelialization was morphologically assessed in the immunohistochemical staining with the anti-CD31 antibody. Reendothelialization was calculated as the ratio of the surface covered by CD31-positive cells to the total luminal surface. The quantification of endothelial staining was performed in a double blind manner by two independent researchers.

2.8 X-gal staining
For the detection of LacZ reporter activity, arterial sections (40 µm in thickness) were fixed in acetone, stained using the X-gal Substrate Set (HistoMark; KPL, Gaithersburg, MD) according to the manufacturer's instructions and carefully examined under a microscope for the presence of blue staining [20].

2.9 Serum cytokine levels
Blood samples were collected after G-CSF or saline was administered for 4 consecutive days prior to vascular injury and 2 days after the injury. The serum levels of interleukin (IL)-6, IL-12p70, tumor necrosis factor-{alpha} (TNF-{alpha}), and monocyte chemoattractant protein-1 (MCP-1) were assessed by using the CBA Mouse Inflammation Kit (BD Biosciences) according to the manufacturer's instructions.

2.10 Statistical analysis
Data are presented as mean±SEM. Student's t test was used to compare two groups. For comparisons between multiple groups, we determined the significance of the difference between group means by one way analysis of variance (ANOVA) combined with Tukey–Kramer's test. All analyses were performed using the StatView software (Abacus Concepts, Inc., Berkeley, CA). A value of p<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effect of G-CSF on neointimal formation and reendothelialization
It has been shown that neointimal formation is complete at 3 or 4 weeks after a wire-mediated vascular injury [19]. Hence, we first evaluated the effect of G-CSF treatment on neointimal formation at 3 weeks after a vascular injury. Histological analysis revealed that the neointimal formation was significantly reduced in the G-CSF-pretreated group when compared with that in the vehicle group (p<0.05; Fig. 1A–D). Although the neointimal formation tended to be reduced in the G-CSF-posttreated group, no significant difference was observed between the G-CSF-posttreated and vehicle groups. Comparative changes were also observed in the luminal area of the injured artery (Fig. 1E). Since the luminal surface of the neointima was almost completely reendothelialized at 3 weeks after the vascular injury, we then determined the ratio of reendothelialization at 1, 3, 5, and 10 days after the injury. Immunohistochemical analysis of the endothelial marker CD31 demonstrated accelerated reendothelialization of the injured artery in the G-CSF-pretreated group when compared with that in the vehicle group (Fig. 2). In the G-CSF-posttreated group, the reendothelialization was slightly improved, but no statistical significance was observed (data not shown).


Figure 1
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  		Fig. 1 Effect of G-CSF treatment on neointimal formation. Recombinant human G-CSF (100 µg/kg/day) was administered from 4 days prior to vascular injury through 6 days after injury (G-CSF-pretreated, n=20) or for 10 days after the injury (G-CSF-posttreated, n=22). Saline was administered for 10 days after the injury (Vehicle, n=22). The femoral arteries were excised 3 weeks after the injury. The sample sections were stained with HE, and neointimal formation was evaluated. (A to C) Representative photographs of HE staining. (A) G-CSF-pretreated, (B) G-CSF-posttreated, and (C) Vehicle. The arrows indicate internal elastic lamina. (D and E) The bar graphs show the area of intima (D) and lumen (E) quantified by NIH Image ver. 1.63. Data are mean±SEM. Bar shows 200 µm.

 

Figure 2
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  		Fig. 2 Effect of G-CSF treatment on reendothelialization. Recombinant human G-CSF (100 µg/kg/day) was administered from 4 days prior to vascular injury through 6 days after injury (G-CSF). Saline was administered for 10 days after the injury (Vehicle). The femoral arteries were excised 1, 3, 5, and 10 days after the injury. Immunohistochemical staining for CD31 was performed and the ratio of reendothelialization was evaluated. (A) Representative photographs of CD31 immunostaining. (B) The bar graph shows the reendothelialization ratio quantified by NIH Image ver. 1.63. Data are mean±SEM (n=3–4). Bar shows 200 µm.

 
3.2 Mobilization of EPCs by G-CSF treatment
To investigate the mobilization of EPCs by G-CSF treatment, the number of EPCs in the peripheral circulation was assessed by flow cytometry 4 days after the start of G-CSF treatment and 2 days after the vascular injury. The number of EPCs, determined by CD34+/Flk1+-expressing cells, was significantly increased in the G-CSF-treated mice when compared with that in the vehicle-treated mice (4.32 vs. 1.60 cells/µL; p<0.05; Fig. 3).


Figure 3
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  		Fig. 3 Mobilization of EPCs by G-CSF treatment. The number of CD34+ (A), Flk-1+ (B), and CD34+/Flk-1+ (C) -expressing cells was assessed by using flow cytometry after G-CSF or saline (Vehicle) was administered for 4 consecutive days and 2 days after the vascular injury. Data are mean±SEM (n=6).

 
3.3 Contribution of bone marrow-derived cells to neointimal formation
To determine the contribution of bone marrow-derived EPCs to the accelerated endothelialization after vascular injury, we used two types of bone marrow-transplantation mice whose bone marrow was replaced with that of GFP-Tg or Tie2/LacZ mice. Since it was difficult to discriminate GFP-expressing cells from other types of cells in the presence of autofluorescence of the injured artery [6], we identified bone marrow-derived cells by immunohistochemical analysis using the anti-GFP antibody. In mice whose bone marrow was replaced with that of GFP-Tg mice (GFP->C57BL/6), many GFP-positive inflammatory cells were detected in the adventitia and smooth muscle cells in the neointima of the injured artery; however, few GFP-positive endothelial cells, determined by CD31 expression on the luminal surface, were observed (Fig. 4A). Few GFP-positive cells were observed in the intact artery of GFP->C57BL/6 mice. As expected, no GFP-positive cells were observed in the injured artery of wild-type mice. Similarly, in the mice whose bone marrow was replaced with that of Tie2/LacZ mice (Tie2/lacZ->FVB), β-galactosidase-positive cells, determined by both X-gal staining and immunohistochemical analysis for β-galactosidase, were rarely detected in the reendothelialized artery (Fig. 4B). Furthermore, no significant differences were observed in the bone marrow-derived EPCs and the smooth muscle cells in the neointima between the G-CSF-treated and vehicle-treated mice (data not shown).


Figure 4
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  		Fig. 4 Contribution of bone marrow-derived cells to neointimal formation. (A) Bone marrow-transplanted mice (GFP->C57BL/6) were developed as described in the Materials and methods. Wire-mediated vascular injury was produced 8 weeks after the bone marrow transplantation. The femoral arteries were excised 3 weeks after the injury, and immunohistochemical staining (IHC) for GFP and CD31 were performed. Few GFP-positive endothelial cells were observed in the luminal surface (arrows). No GFP-positive cells were observed in the intact artery of GFP->C57BL/6 mice and in the injured artery of wild-type mice (C57BL/6). (B) Bone marrow-transplanted mice (Tie2/lacZ->FVB) were developed. Wire-mediated vascular injury was produced 8 weeks after the bone marrow transplantation. The femoral arteries were excised 3 weeks after the injury, and X-gal staining and IHC for β-galactosidase (β-gal) and CD31 were performed. β-galactosidase-positive cells were rarely detected in the reendothelialized artery. Bar shows 200 µm.

 
3.4 Effect of G-CSF treatment on serum cytokine levels
To investigate the other possible mechanisms by which neointimal formation is inhibited by G-CSF treatment, we examined the effect of G-CSF pretreatment and posttreatment on serum cytokine levels. The serum IL-6 levels were significantly decreased in the G-CSF-pretreated mice when compared with those in the vehicle-treated mice (p<0.05; Fig. 5). Although the serum IL-6 level in the G-CSF-posttreated group also tended to be decreased, no statistical significance was observed. Furthermore, no significant differences were observed in the serum IL-12p70, TNF-{alpha}, and MCP-1 levels among these groups (Fig. 5).


Figure 5
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  		Fig. 5 Effects of G-CSF treatment on serum cytokine levels. Blood samples were obtained from the mice in the vehicle, G-CSF-pretreated, or G-CSF-posttreated groups as described in Materials and methods. Serum levels of IL-6 (A), IL-12p70 (B), TNF-{alpha} (C), and MCP-1 (D) were assessed by using CBA mouse inflammatory kit. Data are mean±SEM (n=6).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The major findings of this study are as follows: First, G-CSF pretreatment accelerated reendothelialization and decreased neointimal formation after vascular injury. Second, G-CSF treatment increased the number of EPCs (CD34+/Flk-1+) in the peripheral circulation. Third, few bone marrow-derived EPCs were observed in the reendothelialized artery and these cells were not increased by G-CSF treatment. Finally, G-CSF treatment reduced the serum level of IL-6. These findings provide a new insight into the role of bone marrow-derived cells in the development of neointimal formation after vascular injury and the clinical application of G-CSF to CAD.

There are conflicting reports regarding the effect of G-CSF on neointimal formation after vascular injury. In a small clinical study, Kang et al. [17] showed that a G-CSF injection and an intracoronary infusion of peripheral mobilized stem cells in patients with CAD promoted angiogenesis and improved myocardial perfusion and cardiac function. However, they noted an unexpectedly high rate of in-stent restenosis at the culprit lesion in patients who received G-CSF; therefore, they terminated the study. Conversely, Kong et al. [18] recently reported that G-CSF treatment prior to balloon injury leads to accelerated reendothelialization and inhibition of neointimal formation in the injured arteries in splenectomized rats. They suggested that this approach might be a suitable therapeutic strategy for the prevention of restenosis after revascularization procedures such as percutaneous transluminal angioplasty, stenting, and atherectomy. Consistent with their results, we observed that G-CSF pretreatment accelerated reendothelialization and decreased neointimal formation after vascular injury. In the present study, unlike the study of Kong et al. [18], we further investigated the following three points: (1) the role of bone marrow-derived EPCs; (2) the effect of G-CSF in intact mice without splenectomy; and (3) the effect of G-CSF posttreatment after vascular injury. We used a mouse model of the vascular injury because it has several advantages such as the availability of various antibodies and Tg mice expressing marker genes. In addition, we used CD34+/Flk-1+ as a marker of EPCs in this study. Although CD34 expression appears to be inessential for EPCs [21], CD34+/Flk-1+ was most frequently used as an EPC marker [22–24].

This study demonstrated that bone marrow-derived EPCs contributed slightly to the process of reendothelialization after a vascular injury, although G-CSF treatment augmented EPC mobilization in the peripheral circulation. Askari et al. [25] recently demonstrated that G-CSF treatment alone failed to engraft circulating EPCs into the ischemic myocardium; however, the treatment combined with stromal cell-derived factor (SDF-1) significantly promoted engraftment, suggesting that EPC mobilization alone might be insufficient to induce EPC homing from the peripheral circulation to ischemic or injured tissues. We observed that accelerated reendothelialization and decreased neointimal formation occur on treatment with G-CSF and therefore speculated that the resident endothelial cells at the site of injury might play an important role in the process of reendothelialization. Gulati et al. [26] demonstrated that the EPCs attached to the injured artery induced the proliferation of the neighbouring resident endothelium and accelerated reendothelialization after arterial injury. Furthermore, G-CSF might directly act on endothelial cells as reported by Bussolino et al. [27,28]. They reported that G-CSF stimulates the migration and proliferation of human endothelial cells through the stimulation of the Na+/H+ exchanger and shows definite angiogenic activity [29]. On the other hand, we observed that G-CSF treatment diminished the serum concentration of IL-6, which possesses anti-angiogenic properties [30]. Taken together, our results suggest that G-CSF accelerates reendothelialization through EPC-dependent and EPC-independent pathways. Further investigations are required to elucidate the precise mechanisms of G-CSF-accelerated reendothelialization after vascular injury.

In the present study, we evaluated EPC mobilization, reendothelialization, and neointimal formation in mice without splenectomy and showed that G-CSF treatment was effective even in a non-splenectomized animal. The previous study by Kong et al. [18] was conducted using a splenectomized rat model; they found an approximately 7-fold increase in the number of CD34+ cells. We also observed a marked increase in the number of EPCs induced by G-CSF treatment in splenectomized mice when compared with that in intact mice (data not shown). Since performing a splenectomy in humans is not always feasible, our observations based on a non-splenectomized animal model have potentially important implications for the prevention of restenosis by G-CSF treatment. Another important finding of this study is that pretreatment with G-CSF is more effective in inhibiting neointimal formation than posttreatment with G-CSF. This finding was supported by Kong et al. [18] who reported that G-CSF pretreatment accelerated the ratio of reendothelialization and inhibited neointimal formation after vascular injury. They hypothesized that the rapid reconstitution of the injured endothelium by mobilized EPCs leads to timely restoration of endothelial function and vascular homeostasis, resulting in inhibition of neointimal formation. In addition, we observed that pretreatment with G-CSF more effectively reduced the serum IL-6 concentration, indicating that the timing of G-CSF administration is critical for its efficacy in the enhanced reendothelialization and inhibition of neointimal formation. The dose of G-CSF administration is also important for future clinical application. Because it was reported that the maximal G-CSF doses required for the increase in the number of neutrophils and the inhibition of erythropoiesis are 500 and 250 µg/kg/day, respectively, in mice [31], 100 µg/kg/day of G-CSF was used in this study. Other investigations also used 100–300 µg/kg/day of G-CSF in order to promote EPC mobilization in mice [25,32,33]. Taken together, further studies on the dose and timing of G-CSF administration are required for future clinical applications.

Currently, drug-eluting stents (DES) are widely used in interventional cardiology practice. The introduction of DES has resulted in a significant decrease in the occurrence of restenosis after PCI; however, current therapies with DES interrupt the natural response to vascular injury and impair endothelial repair. The acceleration of the reendothelialization of a vascular injury site after DES implantation has potential clinical benefits in reducing neointimal formation and stent thrombosis. Interestingly, Cho et al. [34] tested the effect of stem cell mobilization by G-CSF on reendothelialization and neointimal formation after DES implantation in rabbits and observed that the combined use of the DES and G-CSF treatments enhanced reendothelialization and inhibited neointimal formation. The proposed combined therapeutic modality of DES and G-CSF merits additional study to confirm synergistic effects on vascular repair and neointima.

In conclusion, the present study demonstrated that G-CSF treatment accelerated reendothelialization and decreased neointimal formation after a wire-mediated vascular injury in mice. Furthermore, it was demonstrated that bone marrow-derived EPCs contributed slightly to the reendothelialization of the artery. Since G-CSF is currently widely used in the treatment of patients with neutropenia as well as for bone marrow reconstitution and stem cell mobilization, these findings suggest that G-CSF pretreatment has a therapeutic potential for the prevention of restenosis after PCI.


   Acknowledgements
 
We thank Junko Yano, Tomoko Hamaji, and Kazuko Misawa for excellent technical assistance and Dr. Masaru Okabe for providing GFP-Tg mice. This study was supported by research grants from the Ministry of Health, Labor and Welfare of Japan (Research on Measures for Intractable Diseases), the Ministry of Education, Science, Sports and Culture, and Daiwa Securities Health Foundation.


   Notes
 
Time for primary review 23 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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