Abstract:Background:We study NF-B-activated CTCF in EGF-induced wound healing. Results: Corneal epithelial wound healing was significantly impaired because of the lack of CTCF activity in NFB-p50 Ϫ/Ϫ mice.
“…In contrast, GO enrichment analysis of the EGF-upregulated genes showed that they were significantly enriched in immune response, response to wounding, and defense response (Table 2). This is concordant with previous reports showing that EGF stimulates CECs during wound healing 13–15, 17 . Our functional analysis indicates that K+Y medium should be suitable for effective ex vivo proliferation of LECs.Figure 3Volcano plot of statistical significance against log 2 fold-change between EGF-treated LECs and K+Y-treated LECs.…”
An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.
“…In contrast, GO enrichment analysis of the EGF-upregulated genes showed that they were significantly enriched in immune response, response to wounding, and defense response (Table 2). This is concordant with previous reports showing that EGF stimulates CECs during wound healing 13–15, 17 . Our functional analysis indicates that K+Y medium should be suitable for effective ex vivo proliferation of LECs.Figure 3Volcano plot of statistical significance against log 2 fold-change between EGF-treated LECs and K+Y-treated LECs.…”
An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.
“…A), both of which have been shown to promote the production of epithelial cells (Singh and Foster, ; Yano et al, ; Qiao et al, ; Richardson et al, ; Wang et al, ; Wong and Rossant, ; Peterson et al, ). For example, EGF plays an important role during the corneal wound healing process by improving epithelial proliferation(Wang et al, ), while KGF markedly influences epithelial growth and differentiation during lung morphogenesis (Shiratori et al, ). The immunofluorescence staining and western blotting results indicated that EGF was able to efficiently induce the expression of CRYAA in the monolayer cells with homogenous morphology (Figs.…”
“…EGFR signaling activates an NF-κB pathway, which leads to transcriptional repressor CTCF activation and PAX6 downregulation (Li and Lu, 2005; Lu et al, 2010; Wang et al, 2012a) resulting in cell migration and proliferation in corneal epithelial cells (Imanishi et al, 2000). Recent studies (Wang et al, 2013) have demonstrated that in NF-κB p50 knockout mice, corneal epithelial wound healing is significantly delayed, suggesting that NF-κB signaling pathways play important roles in growth factor-promoted corneal epithelial self-renewal and wound healing. EGFR signaling also activates histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, and promotes corneal epithelial cell migration and healing of debridement wounds.…”
Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.
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