Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude-nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude-nu, wild-type controls (C57BL/6J), severe-combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude-nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Masson's trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro-scarring cytokines platelet-derived growth factor-B and transforming growth factor beta1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8-positive cells in all studied animals except nude-nu mice. We conclude that scarless skin healing in athymic nude-nu mice provides a new model to study the influence of the immune system on tissue regeneration.

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.

To study the metabolic activity of NF-B, we investigated phenotypes of two different mouse models with elevated NF-B activities. The transcriptional activity of NF-B is enhanced either by overexpression of NF-B p65 (RelA) in aP2-p65 mice or inactivation of NF-B p50 (NF-B1) through gene knock-out. In these models, energy expenditure was elevated in day and night time without a change in locomotion. The mice were resistant to adulthood obesity and diet-induced obesity without reduction in food intake. The adipose tissue growth and adipogenesis were inhibited by the elevated NF-B activity. Peroxisome proliferator-activator receptor ␥ expression was reduced by NF-B at the transcriptional level. The two models exhibited elevated inflammatory cytokines (tumor necrosis factor-␣ and interleukin-6) in adipose tissue and serum. However, insulin sensitivity was not reduced by the inflammation in the mice on a chow diet. On a high fat diet, the mice were protected from insulin resistance. The glucose infusion rate was increased more than 30% in the hyperinsulinemic-euglycemic clamp test. Our data suggest that the transcription factor NF-B promotes energy expenditure and inhibits adipose tissue growth. The two effects lead to prevention of adulthood obesity and dietary obesity. The energy expenditure may lead to disassociation of inflammation with insulin resistance. The study indicates that inflammation may prevent insulin resistance by eliminating lipid accumulation.The IKK␤ 2 /NF-B signaling pathway plays an important role in the control of inflammation, apoptosis, carcinogenesis, and oxidative stress (1). In this pathway, the serine kinase IKK␤ (IKK2) activates the transcription factor NF-B through phosphorylation of NF-B inhibitor (IB␣). In obesity research, the metabolic activity of IKK␤ was tested in the control of insulin sensitivity (2-4) or food intake in transgenic mice (5). In these studies, the IKK␤ activity was modified either globally or tissuespecifically in several major tissues/organs, such as the liver (3, 4), skeletal muscle (6), and brain (5). In these studies, the role of IKK␤ in the regulation of energy expenditure and adipose tissue growth was not examined. Although IKK␤ and NF-B activities are parallel in most cases, their activities are not identical (7). IKK␤ has NF-B-independent activities (7, 8). We investigated the metabolic activity of NF-B using the NF-B transgenic mice in the current study.NF-B activation is associated with energy expenditure in cachexia (9, 10). However, the cause/effect relationship has not been tested for NF-B/energy expenditure in transgenic models. NF-B is a transcription factor that regulates expression of a broad spectrum of genes. Its activity is found in many types of cells, including adipocytes and macrophages (1, 11). The common form of NF-B contains two subunits: p65 (RelA) and p50 (NF-B1). p65 contains the transactivation domain and mediates transcriptional activation of target genes. p50 usually inhibits the transcriptional activity of p65 (12), and the inhibiti...

Similar to mammalian fetuses FOXN1 deficient (nude) mice are able to restore the structure and integrity of injured skin in a scarless healing process by mechanisms independent of the genetic background. Matrix metalloproteinases (MMPs) are required for regular skin wound healing and the distinctive pattern of their expression has been implicated to promote scarless healing. In this study, we analyzed the temporal and spatial expression patterns of these molecules during the incisional skin wounds in adult nude mice. Macroscopic and histological analyses of skin wounds revealed an accelerated wound healing process, minimal granulation tissue formation and markedly diminished scarring in nude mice. Quantitative RT-PCR (Mmp-2,-3,-8,-9,-10,-12,-13,-14 and Timp-1, -2, -3), Western blots (MMP-13) and gelatin zymography (MMP-9) revealed that MMP-9 and MMP-13 showed a unique, bimodal pattern of up-regulation during the early and late phases of wound healing in nude mice. Immunohistochemically MMP-9 and MMP-13 were generally detected in epidermis during the early phase and in dermis during the late (remodeling) phase. Consistent with these in vivo observations, dermal fibroblasts cultured from nude mice expressed higher levels of type I and III collagen, MMP-9 and MMP-13 mRNA levels and higher MMP enzyme activity than wild type controls. Collectively, these finding suggest that the bimodal pattern of MMP-9 and MMP-13 expression during skin repair process in nude mice could be a major component of their ability for scarless healing.

Adipocytes arise from multipotent stem cells of mesodermal origin, which also give rise to the muscle, bone, and cartilage lineages. However, signals and early molecular events that commit multipotent stem cells into the adipocyte lineage are not well established mainly due to lack of an adequate model system. We have identified a novel source of adult stem cells from the external murine ears referred to here as an ear mesenchymal stem cells (EMSC). EMSC have been isolated from several standard and mutant strains of mice. They are self-renewing, clonogenic, and multipotent, since they give rise to osteocytes, chondrocytes, and adipocytes. The in vitro characterization of EMSC indicates very facile adipogenic differentiation. Morphological, histochemical, and molecular analysis after the induction of differentiation showed that EMSC maintain adipogenic potentials up to fifth passage. A comparison of EMSC to the stromal-vascular (S-V) fraction of fat depots, under identical culture conditions (isobutyl-methylxanthine, dexamethasone, and insulin), revealed much more robust and consistent adipogenesis in EMSC than in the S-V fraction. In summary, we show that EMSC can provide a novel, easily obtainable, primary culture model for the study of adipogenesis.

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory effect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.

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After publication of this article, we found an error in Figs. 2E and 5E. Specifically, the figures present energy expenditure results that are normalized with the whole body mass. This is inconsistent with the figure legends, which indicate normalization with the lean body mass. Below, we present the corrected figures to match the figure legends. The difference in normalization does not influence the conclusion based on the figures that energy expenditure is enhanced in aP2-p65 mice on either a chow diet (Fig. 2E) or a high fat diet (Fig. 5E). This correction does not influence the conclusion of the entire study that inflammation is uncoupled with insulin resistance in the transgenic mice. FIGURE 2EFIGURE 5E