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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Effective doses of ionizing radiation during preoperative radiotherapy occasionally cause wound complications after subsequent surgery. The authors attempted to accelerate radiation-impaired wound healing in animal models. Recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human macrophage colony-stimulating factor (rhM-CSF), and an inhibitor of transforming growth factor (TGF)-β1 receptor kinase, SB431542, were injected s.c. into a full-thickness incisional wound site in the dorsal skin of rats after local irradiation of X-ray (30 Gy). Wound healing of irradiated skin was assessed using the breaking strength of the wound and histological analyses. The impaired wound healing in irradiated skin was found to be associated with impaired mobilization of bone marrow-derived cells and enhanced expression of TGF-β1 mRNA. The breaking strength of the wound in the irradiated skin was approximately one-eighth of that in the non-irradiated skin; however, following combined treatment with the above three compounds the breaking strength increased to approximately one-half of that in the non-irradiated skin. Histological analysis of the wounded skin revealed an increase in formation of collagen fibers and the panniculus carnosus following the combined treatment. Moreover, the increased breaking strength was associated with an increase in a subpopulation of fibrocytes (collagen I/ED1 double positive cells). These findings suggested that a combined treatment with rhG-CSF, rhM-CSF, and SB431542 is promising as a means of improving radiation-impaired wound healing. (Cancer Sci 2008; 99: 1021–1028)

Abbreviations:
DMSO

dimethylsulfoxide

GAPDH

glyceraldehyde-3-phosphate dehydrogenese

G-CSF

granulocyte colony-stimulating factor

GFP

green fluorescence protein

Ig

immunoglobulin

M-CSF

macrophage colony-stimulating factor

PI

propidium iodide

rhG-CSF

recombinant human granulocyte colony-stimulating factor

rhM-CSF

recombinant human macrophage colony-stimulating factor

RT-PCR

reverse transcription-polymerase chain reaction

Tg

transgenic

TGF

transforming growth factor

Preoperative radiotherapy is now widely used to treat patients with many types of cancer as a means of reducing the size of the cancer at the primary site and the degree of subclinical local invasion, and the radiation, often together with chemotherapy, has a beneficial effect on patient outcome in various types of cancer, including soft-tissue sarcoma, head and neck cancer, rectal cancer, and esophageal cancer.(1–4) Because effective doses of radiation often damage adjacent normal tissues, wound healing after subsequent surgery is often accompanied by wound complications.(5,6) If proper wound healing could be ensured in such cases, the survival rate and quality of life (QOL) of the patients could be expected to increase.

Vascular endothelial cells and fibroblasts are known to be important targets of ionizing radiation.(7,8) Previous studies have demonstrated impaired wound healing and the development of fibrosis after irradiation in both patients and animal models.(8,9) Although the TGF-b signal is essential for cutaneous wound healing,(10) radiation-induced increases in the TGF-b signal promote fibrosis and retard cutaneous wound healing.(11,12) Wound healing in irradiated skin has been reported to be accelerated in the Smad3-deficient mouse in comparison with the wild-type mouse,(13) suggesting that excessive activation of the TGF-b signal may suppress wound healing in irradiated tissues. SB431542 is a TGF-b1-receptor kinase inhibitor. Because it inhibits TGF-b–Smad3 signal activity in vitro,(14) TGF-b signal inhibitors are candidates for therapeutic agents to suppress the TGF-b signal in vivo.

Several methods for improving wound healing have been reported. Transplantation of normal fibroblasts or bone marrow cells has been shown to slightly accelerate wound healing in irradiated rat skin,(15,16) but chemical treatment has been suggested to be superior to cell transplantation for clinical purposes, because of the difficulty of preparing autologous cells from cancer patients.

Several types of cells are associated with cutaneous wound healing, for example, endothelial cells, fibroblasts, and macrophages. Parts of them are known to originate from bone-marrow-derived precursors.(17–20) G-CSF and M-CSF stimulate the survival, proliferation, and differentiation of the neutrophil lineage and monocyte/macrophage lineage, respectively.(21,22) G-CSF has also been shown to be capable of mobilizing endothelial precursor cells to wound sites(20) and of preventing cardiac ischemia and fibrosis.(23,24) M-CSF has been shown to improve the healing of excisional wounds(25) and to accelerate myocardial infarct repair by accelerating the formation of collagen fibers.(26)

In the present study, the authors attempted to improve wound healing in irradiated rat skin by using rhG-CSF and rhM-CSF to mobilize bone-marrow-derived precursor cells to the wound site and SB431542 to suppress excess activation of the TGF-b signal.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals.  Female 4-week-old Wistar rats and C57BL/6 mice were purchased from SLC Japan and given ad libitum access to food and water. The animal experiments were carried out in accordance with the Guidelines of the Animal Care and Use Committee of the National Cancer Center. The GFP Tg mice used in this study were maintained in the authors’ animal room.(18)

Preparation and wounding of mice transplanted with GFP Tg bone marrow cells.  Transplantation of bone marrow cells was carried out using the method described previously.(18) Bone marrow cells were isolated from the femurs of the GFP Tg mice. After whole-body irradiation (9 Gy), C57BL/6 mice were injected with 1 × 107 of the GFP Tg marrow cells via the tail vein. Four weeks later, both thighs of the mice were irradiated with an electron beam (30 Gy, 7 MeV), and 1 week later a full-thickness excisional wound was created in the skin of the left thigh of the mice with a dispo-punch (4-mm diameter). Seven days after wounding, the non-wounded skin and wounded skin were resected from the right and left thighs, respectively, and examined histologically to detect GFP.

X-ray irradiation and creation of surgical wound.  The entire body of an anesthetized rat was covered with a lead plate (5-mm thick). Part of the dorsal skin (4 × 4 cm) was then pulled out from beneath the lead plate and exposed to X-ray radiation (30 Gy, 150 kV) in a Faxitron Cabinet X-ray System, Model CP-160 (Faxitron X-ray Corporation, USA).

Nine days after irradiation, a full-thickness surgical incision (2.5-cm long), including through the panniculus carnosus, was made in the dorsal skin of irradiated and non-irradiated rats, and all layers of the wounds were closed with 5-0 polypropylene sutures (Keisei Medical Industrial Co. Ltd, Japan).

RT-PCR of TGF-β1 mRNA.  Before and after wounding, total RNA was extracted from the non-irradiated and irradiated skins with an RNA extraction kit (Isogen, Nippon Gene Co. Ltd, Japan). RT-PCR of the total RNA (0.5 µg) was performed with an RNA LA PCR kit (Takara, Japan). The primers used were: 5′-AAACTAAGGCTCGCCAGTCC-3′ (forward) and 5′-GTTGGTATCCAGGGCTCTCC-3′ (reverse) for rat TGF-β1 mRNA, 5′-TCTCCGCCCCTTCCGCTGAT-3′ (forward) and 5′-CCACCACCCTGTTGCTGTAG-3′ (reverse) for rat GAPDH mRNA.

Combined treatment for accelerating wound healing.  rhG-CSF (Neutrogin) and rhM-CSF (Leukoprol) were purchased from Chugai Pharmaceutical Co. Ltd, Japan, and Kyowa Hakko Co. Ltd, Japan, respectively, and dissolved in saline. The rhG-CSF (10 mg/kg/day) and rhM-CSF (300 000 IU/kg/day) were injected s.c. into the center of the irradiated and non-irradiated skin on preoperative days 3, 2, and 1. They were also injected s.c. into one side of the wound on postoperative days 0 and 1. As myeloid hyperplasia has been reported as a function of G-CSF or M-CSF,(27,28) the efficacy of used doses of G-CSF and M-CSF was confirmed by an increase in the number of segmented neutrophils in bone marrow of rats treated with G-CSF and M-CSF (data not shown). SB431542 was purchased from TOCRIS, USA, dissolved in saline containing 50 mmol phosphate-buffer (pH 7.4), 1% DMSO, and 2% Tween 80, and a dose of 500 nmol/kg per day was injected s.c. into the other side of the wound on postoperative days 1, 2, and 3. Groups of four rats each were injected with rhG-CSF alone, rhM-CSF alone, SB431542 alone, rhG-CSF plus rhM-CSF, rhG-CSF plus SB431542, rhM-CSF plus SB431542, rhG-CSF, rhM-CSF plus SB431542, or the solvent alone as a control (n = 8).

Measurement of breaking strength.  On postoperative day 7, wounded skin was excised from the dorsum. A piece of the excised skin (1 cm wide × 3.5 cm long) containing a twisted suture at the center was obtained, and the twisted suture was carefully removed. The breaking strength of the wound was measured with a force gauge (TK-251 A; Muromachi Kikai Co. Ltd, Japan). The remaining pieces of the wounded skin were used for the histological analyses.

Histological examination.  The piece of wounded skin was fixed with 10% buffered formalin and embedded in paraffin. The wound area, including fibrin clots and granulation tissue, was morphologically determined by examining the HE-stained sections. The structure of elastic and collagen fibers was analyzed with the Elastica van Gieson staining.

GFP-positive cells were stained with rabbit polyclonal antibody to GFP (1:500, Molecular Probes, USA), as described previously.(18) For quantitative analysis of the TGF-β signal in frozen sections, nuclear phosphorylated Smad2/3 was detected with a rabbit antibody against phosphorylated Smad 2/3 (1:500, Santa Cruz, USA) and Alexa fluor 488-labeled antirabbit IgG (1:400, Molecular Probes, USA). The nuclei were stained with PI (10 µg/mL), and fluorescence was detected with confocal laser scanning microscope (LSM 5 PASCAL, Carl Zeiss, Germany).

Monocyte/macrophage lineage cells were detected with mouse monoclonal antibody against rat ED1 (1:800, Serotec, USA), and collagen I was detected with rabbit polyclonal antibody against collagen I (1:50, Calbiochem, USA). For double-staining of collagen I/ED1 in the formalin-fixed specimens, after treating the paraffin-embedded sections with 0.1% trypsin (Sigma, USA) for 30 min, the sections were exposed to the primary antibodies for 1 h, followed by Alexa Fluor 488-labeled antirabbit IgG (1:400, Molecular Probes, USA), Alexa Fluor 633-labeled antimouse IgG (1:400, Molecular Probes, USA), and PI (10 µg/mL) for 30 min. Fluorescence was detected with confocal laser scanning microscope (LSM 5 PASCAL, Carl Zeiss, Germany).

Statistical analysis.  The results of the quantitative analyses are expressed as means ± SEM. Differences were considered to be statistically significant at a P-value of 0.05 using Student's t-test. The analyses were performed with StatView ver.5 software on a Macintosh computer.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Regional irradiation inhibits mobilization of bone marrow-derived cells to the skins of GFP–bone marrow cell-transplanted mice.  In order to establish the treatment for improving the wound healing of irradiated skin, the authors tried to find abnormalities in the wound of irradiated skin. First, mobilization of bone-marrow-derived cells was investigated in the irradiated skin of mouse during wound healing. GFP-positive cells were detected in the wounded skin of the GFP bone marrow cell-transplanted mouse (Fig. 1). While the number of GFP-positive cells in the wounded non-irradiated mouse skin increased by 7 days after wounding (Fig. 1a,c,e), the number in the wounded irradiated skin decreased on day 7 (Fig. 1b–e), suggesting impaired recruitment of bone-marrow-derived cells in the wounded irradiated skin of mouse. Thus, the authors decided on the treatment of G-CSF and M-CSF to improve the impaired healing of wounds in irradiated skin of rat.

image

Figure 1. Mobilization of bone marrow-derived cells in wounded skin of mice. Both thighs of green fluorescence protein (GFP) bone marrow cell-transplanted mice were irradiated with X-ray (30 Gy). Nine days later, an excisional wound was created in the left thigh. On day 7 paraffin-embedded sections were made from wounded skin of the left thigh and non-wounded skin of the right thigh. The GFP-positive cells were stained brown immunohistochemically in sections of (A) the non-wounded non-irradiated skin, (B) non-wounded irradiated skin, (C) wounded non-irradiated skin, and (D) wounded irradiated skin. Scale bar = 100 µm. (E) The ratio of GFP-positive cells in the wounded skin to that in non-wounded skin of each mouse (n = 3) was calculated. Note: the numbers of GFP-positive cells differed from mouse to mouse, because their rates of replacement by GFP-bone marrow cells differed.

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Excessive activation of TGF-β1 signal and its suppression by the treatment of SB431542 during wound healing of irradiated rat skin.  Next the expression of TGF-β1 mRNA in the wounded skin of rat was examined using RT-PCR. Experiments on wound healing have been done using a rat model, because the volumes of skin samples obtained from mice were not enough for analyses of wound healing such as histological analysis and breaking strength measurement. The expression of TGF-β1 mRNA increased in the irradiated skin on day 3 after wounding in comparison with the wounded non-irradiated skin (Fig. 2a). Thus, the TGF-β1 signal may be excessively activated during wound healing of irradiated rat skin.

image

Figure 2. Increase in transforming growth factor (TGF)-β1 mRNA expression, and suppression of TGF-β signal by SB431542 treatment in irradiated rat skin. (A) Nine days after irradiation, non-irradiated or irradiated skins of rats were wounded. Total RNA was isolated from these skins on indicated days. The RT-PCR products of TGF-β1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were analyzed by 1.5% agarose gel electrophoresis. (B) Irradiated rat skins were wounded, and treated with or without SB431542 on postoperative days 1, 2, and 3. The wounded skin was removed 45 min after the final injection of SB431542. Phosphorylated Smad2/3 in the nucleus of dermal cells was detected by immunofluorescent staining of frozen sections, and the nuclei were stained with propidium iodide. Red and green signals indicate nuclei and phospho-Smad2/3, respectively. Nuclei of cells activating TGF-β signal appear yellow. Scale bar = 50 µm. (C) The percentage of dermal cells activating TGF-β signal in the wounded skin. More than 200 dermal cells were counted (n = 5).

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The authors investigated whether SB431542 inhibited the TGF-β1 signal in the wounded irradiated skin of rat. Dermal cells that had an activated TGF-β signal were detected by nuclear staining of phosphorylated Smad 2/3 (Fig. 2a,b). Treatment with B431542 decreased the number of cells activating the TGF-β signal in the dermis of wounded irradiated skin to approximately 70% of that in control wounded irradiated skin (Fig. 2c; P = 0.008). Thus, SB431542 suppressed excessive activation of the TGF-β signal in the irradiated skin of rat after wounding. Thus, it was decided to use SB431542 to improve the impaired healing of wounds in irradiated rat skin.

Improvement of breaking strength of wound in the irradiated skin of rat by combined treatment with rhG-CSF, rhM-CSF, and SB431542.  The breaking strength of a wound is a good index of functional recovery during the healing of incisional wounds. The breaking strength of the wound in the irradiated skin of rat was approximately one-eighth (82.5 ± 10.6 g) of that of the wound in the non-irradiated skin (655.0 ± 59.6 g) on day 7 after creation of the incisional wound (Fig. 3a). No improvement in breaking strength was noted when SB431542, rhG-CSF, or rhM-CSF alone was injected at the wound site in the irradiated skin (Fig. 3a,b). The dose of SB431542 used did not reduce the breaking strength of wound in the non-irradiated skin (Fig. 3a). The breaking strength of the wound in the irradiated skin was slightly improved by combined treatment with rhG-CSF and SB431542 (189.5 ± 42.5 g; P = 0.01) in comparison with that in control irradiated skin (92.5 ± 10.6 g; Fig. 3b). Combined treatment with rhG-CSF and rhM-CSF also slightly improved the breaking strength of the wound in the irradiated skin (140.0 ± 14.7 g, P = 0.03) in comparison with control (92.5 ± 10.6 g; Fig. 3b), but the most significant improvement was obtained by combined treatment with rhG-CSF, rhM-CSF, and SB431542 (320.5 ± 49.1 g; P < 0.0001; Fig. 3b). The breaking strength of the wound in the irradiated skin after combined treatment with the three compounds was four-fold that in control irradiated skin, and approximately half that of the wound in the non-irradiated skin. This improvement in breaking strength by the combined treatment with the three compounds was confirmed by triplicate experiments.

image

Figure 3. Accelerated breaking strength of wound in irradiated skin of rat following combined treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human macrophage colony-stimulating factor (rhM-CSF), and SB431542. Nine days after irradiation, a full-thickness incisional wound was created in the dorsal skin of rat. The breaking strength of the wound in the skin was measured on day 7 after wounding. (A) Each group (n = 4) of irradiated or non-irradiated rats was treated with or without SB431542 on postoperative days 1, 2, and 3. (B) rhG-CSF was subcutaneously injected into the wound site of irradiated skins on preoperative days 3, 2, 1 and postoperative days 0 and 1. SB431542 was similarly administered on postoperative days 1, 2, and 3. rhG-CSF, rhM-CSF and SB431542 were injected using the same procedures. The breaking strength was determined using the procedure described in the Material and Methods. Note: P-values with significant differences are: none versus G-CSF, M-CSF, *P = 0.03; none versus G-CSF plus SB431542, *P = 0.01; none versus G-CSF, M-CSF plus SB431542, **P < 0.0001.

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Decrease in the wound area of wounded irradiated rat skin by a combined treatment with rhG-CSF, rhM-CSF and SB431542.  To assess the wound healing morphologically, the wound area, including the fibrin-containing gap and granulation tissue, was measured in HE-stained sections of the wounded rat skin (Fig. 4a,b). The wound area in irradiated skin treated with the combination of rhG-CSF, rhM-CSF, and SB431542 was one-fifth (0.355 ± 0.095 mm2) the area in non-treated irradiated skin (1.532 ± 0.195 mm2) (Fig. 4c), and close to the area (0.175 ± 0.029 mm2) in non-irradiated skin. The decrease in wound area in irradiated skin treated with rhG-CSF and rhM-CSF (1.173 ± 0.212 mm2) was not significant in comparison with the wound area of non-treated irradiated rat skin (Fig. 4c).

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Figure 4. Decrease in the wound area in the irradiated skin following combined treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human macrophage colony-stimulating factor (rhM-CSF), and SB431542. The area of the wound, including the fibrin-containing gap and granulation tissue, was measured in HE-stained sections of irradiated skin following (A) non-treatment or (B) the combined treatment. The areas enclosed by the lines are the wound areas. Scale bar = 100 µm. (C) Four rats per group were analyzed.

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Regeneration of the panniculus carnosus and collagen fibers in the wound of irradiated rat skin following combined treatment.  The width of the cleft in the panniculus carnosus created by incision was measured. The width of the cleft in the wound of irradiated rat skin following combined treatment with rhG-CSF, rhM-CSF, and SB431542 (0.360 ± 0.221 mm) was approximately one-seventh of that in the wound of control irradiated skin (2.045 ± 0.354 mm; Fig. 5a–c), and almost similar to the width in the wound of non-irradiated skin (0.275 ± 0.021 mm), indicating that the combined treatment accelerated regeneration of the panniculus carnosus. No significant decrease in the width of the cleft was found after combined treatment with rhG-CSF and rhM-CSF (1.61 ± 0.398 mm). These findings suggested that regulation of the TGF-β signal is critical to the wound healing of irradiated rat skin.

image

Figure 5. Regeneration of the panniculus carnosus and collagen fibers. The structure of collagen and elastin fibers was detected using Elastica van Gieson staining. The width of the cleft in the broken panniculus carnosus in wounded irradiated skin following (A) non-treatment or (B) the combined treatment was measured. Dark blue lines indicate the widths of the clefts. Scale bar = 1 mm. (C) The widths of the clefts (n = 4) are shown. High-magnification photographs showing the panniculus carnosus and collagen fibers in (D) Elastica van Gieson-stained sections of wounded irradiated skin and (E) wounded irradiated skin following the combined treatment. Large arrows and arrow-heads indicate the front of the regenerating panniculus carnosus and long collagen fibers, respectively. G-CSF, granulocyte colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.

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The Elastica van Gieson staining revealed fragmented collagen fibers in the control wound of irradiated skin (Fig. 5d), whereas structurally normal collagen fibers were found along the regenerating panniculus carnosus in the wound of irradiated rat skin following combined treatment with rhG-CSF, rhM-CSF, and SB431542 (Fig. 5e).

Increase in the number of collagen I/ED1-positive cells in the wound of irradiated rat skin following the combined treatment.  Mobilization of bone-marrow-derived precursor cells was investigated by counting a subpopulation of fibrocytes.(20) Spindle-shaped collagen I/ED1 double-positive cells were found around the regenerating panniculus carnosus (Fig. 6b) in the wound of irradiated rat skin treated with rhG-CSF, rhM-CSF, and SB431542, whereas most of the ED1-positive cells in the wound of the control irradiated skin did not express collagen I (Fig. 6a). The number of collagen I/ED1 double-positive cells was higher in the wound of irradiated skin treated with rhG-CSF, rhM-CSF, and SB431542 (75.7 ± 9.1) than in the wound of irradiated skin treated with solvent alone (16.7 ± 3.8) or with rhG-CSF and rhM-CSF (31.7 ± 0.9) (Fig. 6c). Large numbers of collagen I/ED1 double-positive cells were also found in the wound of non-irradiated skin (104.0 ± 6.8). These findings suggest that the improvement in the breaking strength of the wound in the irradiated skin of the rat following combined treatment is attributable to regeneration of collagen fibers and the panniculus carnosus, both of which were associated with an increase in the number of collagen I/ED1 double-positive cells.

image

Figure 6. Increase in the number of collagen I/ED1 double-positive cells following the combined treatment. Irradiation and creation of the incisional wound were performed using the methods described in Materials and Methods. Collagen I (green) and ED1 (red) double-positive cells were identified using confocal laser microscopy of formalin-fixed sections. The nuclei were stained with propidium iodide and appear blue. The cytoplasm in collagen I/ED1 positive cells is stained yellow. *Collagen I/ED1 double-positive cell. Scale bar = 10 µm. (A–C) Collagen I and/or ED1 double-positive cells in wounded irradiated skins following (A) non-treatment, (B) the combined treatment, or (C) wounded non-irradiated skin. (D) The numbers of collagen I/ED1 double-positive cells in the sections (10 mm2) were calculated. G-CSF, granulocyte colony-stimulating factor; M-CSF, macrophage colony-stimulating factor.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Several attempts to improve radiation-impaired wound healing have been reported. There have been no reports of compounds that are capable of significantly accelerating wound healing in irradiated skin. In an attempt to greatly accelerate wound healing in irradiated skin in the present study, the authors treated the wound with a combination of rhG-CSF, rhM-CSF, and an inhibitor of TGF-β1 receptor kinase, SB431542. The results clearly indicated that the combined treatment of these three compounds successfully improved radiation-impaired wound healing in the rat skin model.

The mechanisms of cutaneous wound healing are very complicated, and numerous factors play a role.(10) A previous report has indicated that exogeneous TGF-β1 accelerated the closure of superficial excisional wounds in normal mouse skin, but suppressed healing of full-thickness excisional wounds.(29) Regeneration of the epidermis may be dependent on the TGF-β signal; however, ionizing radiation has been shown to promote fibrosis in the lung and skin by enhancing the TGF-β signal and to impair wound healing.(11,12) The radiation-induced impairment of wound healing was attenuated in Smad3-knockout mice.(12,30) These findings suggest that radiation-induced impairment of wound healing is at least partly attributable to excessive activation of the TGF-β signal. The results of RT-PCR in the present study showed that TGF-β1 mRNA expression increased on day 3 after wounding in the irradiated skin of rat (Fig. 2a). Because phosphorylation of Smad2/3 on postoperative day 3 was partially suppressed by SB431542 (Fig. 2c), consecutive treatment with SB431542 on postoperative days 1, 2, and 3 was probably effective in suppressing excess activation of the TGF-β1 signal of irradiated rat skin. In addition, because the dose of SB431542 used did not reduce the breaking strength of wound in non-irradiated skin (Fig. 3a), partial inhibition of the TGF-β1 signal in the early stage of wound healing may contribute to accelerating the improvement in the breaking strength of the wound in irradiated rat skin. The breaking strength of the wound in irradiated skin recovered to half of that in non-irradiated skin following the combined treatment in the present study.

It is known that fibroblasts are mobilized from the dermis adjacent to the wound.(31) Moreover, they differentiate from peripheral blood precursors at the wound site.(17,32) Bone-marrow-derived fibroblast precursors, namely fibrocytes, express hematopoietic markers, myeloid antigens, and fibroblast products, including CD45, CD11b, CD13, ED1, and collagen I. G-CSF is a novel factor that mobilizes several precursor cells of fibroblast and endothelial cell from the bone marrow.(20) M-CSF increases the number of tissue macrophages.(32) The number of collagen I/ED1-positive cells increased in the wound site of irradiated rat skin following combined treatment with rhG-CSF, rhM-CSF, and SB431542. Because combined treatment with rhG-CSF and rhM-CSF hardly enhanced the mobilization of collagen I/ED1-positive cells, SB431542 may raise the environment capable for homing of the precursor cells in the wounded skins of rats.

G-CSF and M-CSF are expressed in the inflamed wound sites during cutaneous wound healing.(10) In the present study, the RT-PCR of these cytokine mRNA showed that expression of these mRNA was not enhanced by the combined treatment of the cytokines and inhibitor on day 7 after wounding (data not shown). Probably, local expression of these mRNA was depend on inflammation, but not related to the late stage of wound healing in the skins treated with exogenous G-CSF and M-CSF.

Although it was predicted that the combined treatment might also stimulate angiogenesis in the wound site of irradiated rat skin, no significant increase in the number of factor-VIII-related antigen-positive vessels was found (data not shown). The improvement in the breaking strength of the wound in the irradiated rat skin following the combined treatment was also associated with a decrease in wound area (Fig. 4), structurally normal collagen fibers, and regeneration of the panniculus carnosus (Fig. 5). These findings suggested that the combined treatment promoted scarless healing of the cutaneous wounds.

The Ki-67 positive cells in collagen I-positive cells were hardly detected in the wounded skins of rats after the combined treatment (data not shown). Thus an increase in mobilization of collagen I/ED1-double positive cells is probably not associated with their proliferation in the impaired wound healing of irradiated rat skin.

From the authors’ previous study using GFP bone marrow cell-transplanted mice,(19) it was found that most of mobilized inflammatory cells were bone-marrow-derived cells in wounded skins of the GFP-tagged mice. As mobilization of bone-marrow-derived cells may also occur during wound healing of rat skin, the collagen I/ED1-double positive cells may be derived from bone marrow cells in the present study. The enhanced mobilization of the collagen I/ED1-double positive cells by the combined treatment might be confirmed by an experiment of bone marrow transplantation using GFP bone marrow cell-transplanted rats.

In addition, combined treatment, including changes in the doses of these compounds, should be modified to restore wound healing in irradiated skin to the normal level found in non-irradiated skin.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports, and Culture, Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References