Real-time monitoring of keratin 5 expression during burn re-epithelialization.
ABSTRACT Keratin is a major protein produced during epithelialization following burn injury and is a useful marker for assessing wound healing. Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the keratin 5 (K5) promoter (K5GFP mice) were used to monitor keratin expression, and thus, re-epithelialization of burn wounds.
K5GFP transgenic mice were created using conventional techniques, with PCR and Southern blot confirmation of transgene incorporation, followed by selection of the line with the most intense and consistent basal epithelial EGFP expression. Epi-fluorescent microscopy of 24 K5GFP mouse flanks and 10 negative littermate controls was used to characterize EGFP intensity, before wounding and serially for 30 days after administration of a standardized burn wound and excision. Biopsy sections of K5GFP and negative control mice were stained with K5 antibody and imaged with confocal microscopy to characterize the distribution of EGFP and K5 at baseline and after injury and to examine the correlation between K5 expression and EGFP expression during healing.
Green fluorescence intensity increased at the advancing wound margin of burned K5GFP mice, reaching a maximum between days 12 and 15 post-burn and then decreasing as healing completed. K5 and EGFP expression increased in parallel in burned K5GFP mice as demonstrated by confocal microscopy.
EGFP expression correlated with K5 expression during wound healing and therefore serves as a good marker of re-epithelialization. This transgenic model allows noninvasive, real-time assessment of in vivo K5 expression and will be useful in the study of wound healing.
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ABSTRACT: Significant damage to tissue surrounding burn injuries occurs after the removal of the thermal source. This damage is caused by a combination of both necrotic and apoptotic cell death in the zone of stasis. Preserving the zone of stasis can reduce the wound size and thereby improve wound healing. We tested whether a peptide previously identified to inhibit necrotic and apoptotic cell death in neurons through c-Jun inhibition could enhance wound healing. We first tested the effects of this peptide on a keratinocyte and fibroblast cell line in culture. The peptide promoted proliferation of keratinocytes but had no effect on fibroblast proliferation, while the peptide also inhibited ultraviolet-induced apoptosis of keratinocytes. We finally tested the peptide in vivo, using a mouse model of burn injury. Wounds that were treated with the peptide reepithelialized faster than controls, while cell death surrounding the wound site was markedly reduced 24 hours postinjury, suggesting that the prevention of apoptosis as well as the proliferative effects of this peptide contribute to the wound healing process. Our data implicate c-Jun in multiple processes during wound repair and demonstrate that treatment of burn injuries using inhibitors of c-Jun dimerization at the time of injury can promote wound healing.Wound Repair and Regeneration 12/2007; 16(1):58-64. · 2.76 Impact Factor
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ABSTRACT: Convincing evidence of epithelial damage and aberrant repair exists in adult asthmatic airways, even in the absence of inflammation. However, comparable studies in children have been limited by access and availability of clinical samples. To determine whether bronchial epithelial cells from children with asthma are inherently distinct from those obtained from children without asthma. Epithelial cells were obtained by nonbronchoscopic bronchial brushing of children with mild asthma (n = 7), atopic children without asthma (n = 9), and healthy children (n = 12). Cells were subject to morphologic, biochemical, molecular, and functional assessment. Responses were also compared with commercially available epithelial cultures and the transformed cell line 16HBE140. All epithelial cells exhibited a "cobblestone" morphology, which was maintained throughout culture and repeated passage. Expression of cytokeratin 19 varied, with disease phenotype being greatest in healthy nonatopics and lowest in asthmatics. In contrast, expression of cytokeratin 5/14 was greatest in asthmatic samples and least in healthy nonatopic samples. Asthmatic epithelial cells also spontaneously produced significantly greater amounts of interleukin (IL)-6, prostaglandin E2, and epidermal growth factor, and equivalent amounts of IL-1beta and soluble intracellular adhesion molecule-1, but significantly lower amounts of transforming growth factor beta1. This profile was maintained through successive passages. Asthmatic epithelial cells also exhibited greater rates of proliferation than nonasthmatic cells. This study has shown that epithelial cells from children with mild asthma are intrinsically different both biochemically and functionally compared with epithelial cells from children without asthma. Importantly, these differences are maintained over successive passages, suggesting that they are not dependent on an in vivo environment.American Journal of Respiratory and Critical Care Medicine 12/2006; 174(10):1110-8. · 11.04 Impact Factor