The Effects Of KGF Knockout In Wound Healing

Objectives:Growth factors play an important role in the wound healing process, acting as triggers to stimulate sedentary cell lineages to proliferate and ultimately produce new cells at the wound site. Skin is a complex organ, which consists of a variety of different cell types. A series of intricate and intertwined intercellular interactions are involved in coordinating and maintaining the appropriate proportions and differentiation states of the cell types within the skin. Various types of growth factors have been discovered at the wound site. Keratinocyte growth factor (KGF) is the seventh member of fibroblast growth factor (FGF) family, so it is also known as FGF-7. KGF is a paracrine growth factor that is synthesized by various stromal cell types including fibroblasts, endothelial cells, smooth-muscle cells, and dendritic epidermal T cells. It has been shown that KGF specifically signals through a surface receptor FGFR2-IIIb, which is expressed predominantly by epithelial cells. KGF was considered as a paracrine mediator owing to its expression by mesenchymal cells and mitogenic activity on epithelial cells. However, the activity of KGF on mesenchymal cells was not well explored in vivo. The expression of FGF-7 increased 160 times after cutaneous injury in mouse and human full-thickness excisional wounds. KGF can stimulates migration and multiplication of keratinocytes in vitro. The stimulating effects of KGF on DNA synthesis in cultured keratinocytes are 2-10 times greater than TGF-αor EGF. In addition, the introduction of exogenous KGF to wounded skin can exert an important effect on re-epithelialization. These findings indicate that FGF-7 plays an important role during wound repair. Thus, we created KGF null mice by ablating the KGF gene and observed the developement and phenotype of the mice. We explore the effects of lacking FGF-7 on wound healing by generating full-thickness excisional dorsal wounds in KGF null mice. With the growing number of diabetic population in the world, the complications of diabetes mellitus, such as retinopathy, diabetic nephropathy, neuropathy and chronic wounds are threatening these patients. Diabetic patients with minor wounds often lead to chronic, non-healing ulcers that are predisposed to infection. It is not uncommon for the infection to lead to gangrene and, ultimately, the need for amputation, which has became a great problem that the clinicians are facing. In genetically diabetic mice, wound healing impairment is reflected as prolonged inflammation, impaired neovascularization and decreased synthesis of collagen. Expression profiles of various growth factors are also aberrant in diabetic mice. Wound healing in diabetic mice is remarkably delayed, but can be reversed by the topical application of growth factors such as platelet-derived growth factor (PDGF) and several members of fibroblast factor family. In this study, we explore the effects of lacking FGF-7 on diabetic wound healing by generating full-thickness excisional dorsal wounds in KGF null diabetic mice.Methods:1. The KGF gene consists of three exons and two introns, in which sequences of exon 1 have been shown to be indispensable. Guo from our lab used embryonic stem (ES) cell technology to ablate the KGF gene in mice by silencing exon 1 and to create KGF knockout mice. KGF knockout mice supply us a tool to study the effects of KGF in mice. In order to explore the effects of KGF in diabetic mice, we developed an animal model of diabetic mice that lack FGF-7 by hybridization. We crossed the male FGF-7 null mice (FGF-7-/- Lepr+/+) with the female heterozygous diabetic mice (FGF-7+/+ Leprdb/+) to generate breeders. Those with Leprdb/+ genotype were selected and crossed with each other. The genotypes of their progeny were identified by southern blot and PCR, and the progeny were assigned to Exp. (FGF-7-/- Leprdb/db), Db/db (FGF-7+/+ Leprdb/db), FGF-7 null (FGF-7-/- Lepr+/+,) and WT (FGF-7+/+ Lepr+/+,) according to respective genotype. The phenotypes of the 4 groups were observed.2. Full-thickness excisional dorsal wounds were created in KGF null mice (KGF null, n=12) and wild type mice (WT, n=12). Wound closure rates were measured. Wound tissues were harvested on day 7 for immunohistochemical staining to investigate wound re-epithelialization (HE), cell proliferation (Ki-67) and wound vascularization (CD-31). Realtime-PCR was determined for Col-Ⅰ,α-SMA, TGF-β, bFGF, EGF, IGF-1, and VEGF expression.3. Full-thickness excisional dorsal wounds were created in KGF null diabetic mice (Exp., n=8) and diabetic mice (Db/db, n=8). Wound closure rates were measured. Wound tissues were harvested on day 7 for immunohistochemical staining to investigate wound re-epithelialization (HE), cell proliferation (Ki-67) and vascularization (CD-31). Realtime-PCR was determined for Col-I, a-SMA, TGF-p 1, bFGF, EGF, IGF-1, and VEGF expression.Results:1. KGF null mice developed a matted and greasy appearance. The hair follicle in KGF null mice aligned in a disorganized order. The KGF null diabetic mice and diabetic mice exhibited heavier body weights compared to KGF null mice or wild type mice. Moreover, the body weights of KGF null diabetic mice appear slightly less than those of diabetic mice. This type of mouse developed a phenotype of obesity which resembles diabetic mice, as well as matted and greasy coat which resembles the FGF-7 null mice.2. Lacking KGF resulted in no differences between KGF null and WT in wound closure. KGF null showed decreased proliferation of keratinocytes, whereas the epithelium migration and epithelialization area displayed no statistical difference compared with WT. KGF null also demonstrated decreased angiogenesis and VEGF expression, however, no difference were shown in the expression of Col-I, a-SMA, TGF-β1, bFGF, EGF, IGF-1 between KGF null and WT.3. Lacking KGF delayed wound healing in Exp. Compared with Db/db. The contraction rate in Exp. was significantly lower than that in Db/db, while the epithelialization rate in Exp. was comparable to that in Db/db. The two groups showed no difference in proliferation of keratinocytes, epithelium migration, epithelialization area and angiogenesis. Realtime-PCR expression of growth factors including TGF-β1, bFGF, EGF and VEGF in Exp. was also comparable with Db/db.Conclusion:1. KGF knockout appeared to affect the development of hair follicle.The KGF null diabetic mice developed a phenotype of obesity, which resembles diabetic mice, as well as matted and greasy coat, which resembles the KGF null mice.2. Lack of KGF in normal mice did not affect wound healing. The decreased proliferation of keratinocytes and angiogenesis did not result in wound closure differences. However, we have demonstrated that lack of KGF induced decreased VEGF expression and angiogenesis in vivo, which suggests that KGF plays a role in regulation of VEGF expression and angiogenesis during wound healing.3. Lack of KGF significantly delayed wound healing in diabetic mice as a result of reduced wound contraction rate by further altering the dermal components in diabetic mice.