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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)
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.
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- Materials and Methods
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.