The role of stem cell factor and c-KIT in keloid pathogenesis: do tyrosine kinase inhibitors have a potential therapeutic role?

BACKGROUND: Keloids are fibroproliferative disorders characterized by increased deposition of extracellular matrix components. Stem cell factor (SCF) and its receptor c-KIT are expressed in a wide variety of cells and have also been demonstrated to be important modulators of the wound healing process. OBJECTIVES: To examine the role of the SCF/c-KIT system in keloid pathogenesis. METHODS: Immunohistochemical staining and Western blot analyses were used to examine localization and expression of SCF and c-KIT in keloid and normal skin tissue. This was followed by the detection of SCF and c-KIT expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were cocultured with the fibroblasts. SCF and c-KIT expression levels in all cell extracts and conditioned media were assayed by Western blotting. In another set of experiments, the effect of imatinib (Glivec(®), Gleevec(®); Novartis Pharma AG, Basel, Switzerland) on keloid fibroblasts was examined. RESULTS: SCF and c-KIT were upregulated in keloid scar tissue and in cultured fibroblasts stimulated with serum, highlighting their importance in the initial phase of wound healing. We further demonstrated that epithelial-mesenchymal interactions, mimicked by coculture of keratinocytes and fibroblasts in vitro, not only stimulated secretion of the soluble form of SCF in keloid cocultures but also brought about shedding of the extracellular domain of c-KIT perhaps by upregulation of tumour necrosis factor-α converting enzyme which was also upregulated in keloid scars in vivo and keloid cocultures in vitro. In addition keloid cocultures expressed increased levels of phosphorylated c-KIT highlighting an activation of the SCF/c-KIT system. Finally, we demonstrated that imatinib, a tyrosine kinase inhibitor, may be a possible therapeutic agent for keloids. CONCLUSION: These data indicate that the SCF/c-KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.

Hepatoma-derived growth factor and its role in keloid pathogenesis.

Hepatoma-derived growth factor (HDGF) is a novel mitogenic growth factor that has been implicated in many different carcinomas. Its role in keloid biology has not yet been investigated. The present study is aimed at examining the role of HDGF in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine in vivo localization and expression of HDGF in keloid and normal skin tissue. This was followed by the detection of HDGF expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were co-cultured with the fibroblasts. HDGF expression levels in all cell extracts and conditioned media were assayed through Western blot analysis. In another set of experiments, the effect of exogenous recombinant HDGF on keloid fibroblasts (KF) and normal fibroblasts (NF) was examined. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by quantifying proliferating cell nuclear antigen (PCNA) expression. Downstream targets of HDGF were identified by detecting their expression through Western blot analysis. Our results indicate that there was an increase in HDGF expression in the dermis of keloid compared with normal skin tissue. The application of serum and epithelial-mesenchymal interactions did not seem to have any effect on intracellular HDGF expression levels. However, co-culturing keloid keratinocytes with KFs resulted in increased HDGF secretion when compared with monoculture or normal controls. Furthermore, treatment with exogenous recombinant HDGF was found to increase the proliferation of KFs, activate the extracellular signal-regulated kinase (ERK) pathway and up-regulate the secretion of vascular endothelial growth factor (VEGF).

Cord lining progenitor cells: potential in vitro adipogenesis model.

OBJECTIVE: To investigate the effect of glucose and insulin concentrations on differentiation of umbilical cord lining progenitor cells to adipocyte-like cells (ALCs). METHODS: Cord lining mesenchymal cells (CLMCs) were isolated from the explant of human umbilical cord amniotic membrane. CLMCs were subjected to differentiation under various culture conditions for 20 days. Lipid droplets were confirmed with Oil Red O staining. Gene expressions of adipsin and peroxisome proliferator-activated receptor gamma (PPARγ) were analyzed using reverse transcription-PCR. Leptin and adiponectin secretions were detected using enzyme-linked immunosorbent assay kit. RESULTS: CLMCs became irregular, cuboidal-shaped cells that resemble adipocytes, and Oil Red O staining showed the presence of lipid droplets. The gene expressions of PPARγ and adipsin were upregulated. Leptin and adiponectin secretions by naive CLMCs were below the limits of detection. Matured ALCs cultured in low-glucose medium significantly secreted leptin and adiponectin, whereas those in high-glucose medium significantly secreted only leptin. Insulin concentration affects leptin but not adiponectin secretion. CONCLUSIONS: Under different culture conditions, CLMCs can differentiate into ALCs that resemble adipocytes in either normal-weight or obese individuals. Hence, these ALCs have the potential to be used as an in vitro model to study adipogenesis and obesity, and possibly as a drug discovery model for metabolic disorders.

Comparative proteomic analysis between normal skin and keloid scar.

BACKGROUND: Keloids are pathological scars and, despite numerous available treatment modalities, continue to plague physicians and patients. OBJECTIVES: Identification of molecular mediators that contribute to this fibrotic phenotype. METHODS: Two-dimensional gel electrophoresis, MALDI-TOF, Mascot online database searching algorithm and Melanie 5 gel analysis software were employed for comparative proteomic analysis between normal skin (NS) and keloid scar (KS) tissue extracts. RESULTS: Seventy-nine protein spots corresponding to 23 and 32 differentially expressed proteins were identified in NS and KS, respectively. Isoforms of heat shock proteins, gelsolin, carbonic anhydrase and notably keratin 10 were strongly expressed in NS along with manganese superoxide dismutase, immune components, antitrypsin, prostatic binding protein and crystalline. Various classes of proteins were found either to be present or to be upregulated in keloid tissue: (i) inflammatory/differentiated keratinocyte markers: S100 proteins, peroxiredoxin I; (ii) wound healing proteins: gelsolin-like capping protein; (iii) fibrogenetic proteins: mast cell ß-tryptase, macrophage migration inhibitory factor (MIF); (iv) antifibrotic proteins: asporin; (v) tumour suppressor proteins: stratifin, galectin-1, maspin; and (vi) antiangiogenic proteins: pigment epithelium-derived factor. Significant increases in expression of asporin, stratifin, galectin-1 and MIF were observed by Western blot analysis in KS. CONCLUSIONS: This work has identified differentially expressed proteins specific to KS tissue extracts which can potentially be used as specific targets for therapeutic intervention.