Chemokine receptor CCR7 is expressed in muscle fibers in juvenile dermatomyositis

To better elucidate the pathogenesis of lymphocyte recruitment of memory CD4(+) T cells in inflammatory myopathies, we studied the expression of CCR5 and CCR7 on CD4 memory T cells in muscle tissue from 11 patients with juvenile dermatomyositis, six adult patients with polymyositis, two patients with Duchenne muscular dystrophy, and two patients with spinal muscular atrophy. A prevalent infiltration of CCR5(+) effector CD4 T memory cells is observed in inflammatory myopathies. Moreover, we found a strong expression of CCR7 in perifascicular atrophic and in degenerating/regenerating muscle fibers in juvenile dermatomyositis (JDM) but not in fibers from adult polymyositis and Duchenne muscular dystrophy. The selective expression of CCR7 in JDM may open new perspectives in the understanding of the pathogenesis of inflammatory myopathies, offering a new tool for the differential diagnosis of these disorders.     

Chemokine CCL20 enhances the growth of HuH7 cells via phosphorylation of p44/42 MAPK in vitro

CCR6 is the receptor of chemokine CCL20. In the present study, we demonstrated that the surface expression of CCR6 was enhanced on the human HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially on HuH7 cells, but not on HLE or HLF cells. These HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially the HuH7 cells secreted a significant amount of CCL20 spontaneously, whereas HLE or HLF did not. Stimulation by CCL20 up-regulated the mRNA expression of CCR6 in HuH7 cells and significantly enhanced the growth of HuH7 cells. CCL20-stimulated growth of HuH7 cells was abrogated by the inhibition of downstream signal transduction pathway mediated by p44/42 MAPK, but not by p38 MAPK or SAPK/JNK. CCR6 expression in human HCC tissues was confirmed by RT-PCR. These results indicate that the growth of a proportion of human HCC cells may be mediated by CCL20-CCR6 axis, like HuH7 cells, in an autocrine or paracrine manner.     

Quantitative analysis of epithelial changes during wound healing in palatal mucosa of guinea pigs

Summary The epithelium of wounded guinea pig palate was subjected to stereologic analysis. A total of 18 biopsies (animals) were used. Biopsies were taken at 18, 48, 96 and 120 h after wounding. Point counting procedures were employed to analyse electron micrographs sampled from one (18 h) or two epithelial strata (48, 96 and 120 h). The essential modulations in epithelial structure as wound healing proceeds were as follows: During the early phases characterized by formation and advancement of epithelial lips (18 and 48 h), migrating cells converged towards a cell type which structurally was less differentiated than normal basal cells. This alteration was expressed by a decrease in volume density of cytoplasmic organelles, mainly mitochondria, free ribosomes and tonofilament bundles, coupled with an increase in volume density of lysosomal bodies. Concomitantly, the volume density of cytoplasmic ground substance rose markedly. Subsequent to fusion of contralateral migratory lips (96 and 120 h) reversion to normal epithelial structure was indicated by the increment in magnitude of basal cell parameters. Further structural density gradients from basal towards upper cell layers appeared. This pattern was mainly displayed by mitochondria, free ribosomes, and tonofilament bundles. The magnitude and gradation of most tissue and cell parameters were not yet re-established at 120 h. The density of tonofilament bundles and the density level of cytoplasmic ground substance in particular deviated.This investigation was supported in part by grants No. 512-5958 and No. 512-5151 from the Danish State Medical Research Council     

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.     

Cellular and molecular facets of keratinocyte reepithelization during wound healing

Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.     

Antler stem cells and their potential in wound healing and bone regeneration

Compared to other vertebrates, the regenerative capacity of appendages in mammals is very limited. Deer antlers are an exception and can fully regenerate annually in postnatal mammals. This process is initiated by the antler stem cells (AnSCs). AnSCs can be divided into three types: (1) Antlerogenic periosteum cells (for initial pedicle and first antler formation); (2) Pedicle periosteum cells (for annual antler regeneration); and (3) Reserve mesenchyme cells (RMCs) (for rapid antler growth). Previous studies have demonstrated that AnSCs express both classic mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs), and are able to differentiate into multiple cell types in vitro. Thus, AnSCs were defined as MSCs, but with partial ESC attributes. Near-perfect generative wound healing can naturally occur in deer, and wound healing can be achieved by the direct injection of AnSCs or topical application of conditioned medium of AnSCs in rats. In addition, in rabbits, the use of both implants with AnSCs and cell-free preparations derived from AnSCs can stimulate osteogenesis and repair defects of bone. A more comprehensive understanding of AnSCs will lay the foundation for developing an effective clinical therapy for wound healing and bone repair.     

Interleukin (IL)-19 promoted skin wound healing by increasing fibroblast keratinocyte growth factor expression

BackgroundInterleukin (IL)-19, a member of the IL-10 cytokine family, is involved in keratinocyte proliferation in psoriasis.ObjectivesWe investigated the role of IL-19 in the wound-healing process in vivo and in vitro.MethodsTwo full-thickness circular wounds (4 mm in diameter) were punched into the skin of BALB/C mice. IL-19 and keratinocyte growth factor (KGF) mRNA in wounded skin were determined using real-time PCR. The wounds were treated with PBS, vehicle, IL-19 (400 ng/mL), or IL-20 (400 ng/mL) (n = 6 in each group) twice daily and the percentage of wound healing was measured daily for 7 days. In vitro, human skin fibroblast CCD966-SK cells and keratinocyte HaCaT cells were treated with IL-19 or KGF. Cell proliferation and migration were determined using bromodeoxyuridine (BrdU) and transwell assays, respectively. The expression of IL-19 and KGF mRNA was also analyzed.ResultsIn wounded mouse skin, IL-19 mRNA was upregulated at 12 h, and KGF at 24 h after the injury. Both increases in gene expression declined 72 h after the skin had been wounded. The percentage of wound healing in IL-19-treated mice was higher than in control mice. In vitro, IL-19 upregulated KGF expression in the CCD966-SK cells; IL-19 was upregulated in KGF-treated HaCaT cells. KGF but not IL-19 promoted HaCaT cell proliferation. However, IL-19 significantly increased the migration of HaCaT cells. HaCaT cells treated with the cultured supernatants of IL-19-stimulated CCD966-SK cells showed significantly more proliferation than in controls.ConclusionsIL-19 is important for cutaneous wound healing because it upregulates KGF expression.     

Dynamic changes in connexin expression correlate with key events in the wound healing process

Wound healing is a complex process requiring communication for the precise co-ordination of different cell types. The role of extracellular communication through growth factors in the wound healing process has been extensively documented, but the role of direct intercellular communication via gap junctions has scarcely been investigated. We have examined the dynamics of gap junction protein (Connexins 26, 30, 31.1 and 43) expression in the murine epidermis and dermis during wound healing, and we show that connexin expression is extremely plastic between 6 hours and 12 days post-wounding. The immediate response (6 h) to wounding is to downregulate all connexins in the epidermis, but thereafter the expression profile of each connexin changes dramatically. Here, we correlate the changing patterns of connexin expression with key events in the wound healing process.     

Rho-family small GTPases are required for cell polarization and directional sensing in Drosophila wound healing

A wound induces cell polarization, in which myosin II is localized at the rear end of individual cells in a migrating epithelial sheet of the Drosophila larval epidermis. Here, we use myosin localization to demonstrate that Rac1, Cdc42, and Rho1 are each required for cell polarization and directional sensing of the wound. The three GTPases are also required for actin cable formation at the wound leading edge. Rac1, Cdc42, and Rho1 act upstream of c-Jun N-terminal kinase (JNK) to organize actin assembly. These results highlight the similarities between the molecular mechanism of Drosophila wound healing and those of Drosophila embryonic dorsal closure and the chemotactic response of Dictyostelium and leukocytes.     

VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing

Vascular endothelial growth factor (VEGF) receptor activation regulates endothelial cell (EC) survival, migration and proliferation. Recently, it was suggested the cross-talk between the VEGF receptors-1 (FLT-1) and -2 (KDR) modulated several of these functions, but the detailed molecular basis for such interactions remained unexplained. Here we demonstrate for the first time that VEGF stimulation of EC monolayers induced a rapid FLT-1-mediated internalization of KDR to the nucleus, via microtubules and the endocytic pathway, internalization which required the activation of PI 3-kinase/AKT. KDR deletion mutants were generated in several tyrosine residues; in these, VEGF-induced KDR internalization was impaired, demonstrating this process required activation (phosphorylation) of the receptor. Furthermore, we demonstrate that in vitro wounding of EC monolayers leads to a rapid and transient internalization of VEGF+KDR to the nucleus, which is essential for monolayer recovery. Notably, FLT-1 blockade impedes VEGF and KDR activation and internalization, blocking endothelial monolayer recovery. Our data reveal a previously unrecognized mechanism induced by VEGF on EC, which regulates EC recovery following wounding, and as such indicate novel targets for therapeutic intervention.     

Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing

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Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice

Cutaneous wound healing is associated with migratory and remodeling events that require the action of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Differences in their expressions were observed during scar-forming and scar-free skin wound healing. We previously found that athymic nude mice are exceptional among mature mammals in their ability to heal injured skin scarlessly. The present study was undertaken to determine whether the modulation of MMP-2 and MMP-9 expression during scarless healing in nude mice was different from scar-forming animals. Full thickness skin wounds were made into the back of nude, wild-type controls (C57BL/6J), immunodeficient SCID and Rag, thymectomized neonates and adults, and cyclosporin A treated mice. Post-injured skin tissues were harvested at Day 7 and 24 after injury. Quantitative RT-PCR, Western blot, gelatin zymography and immunohistochemical assays were performed. Our results show that MMP-2 protein was high but similarly expressed in all post-injured animals on Day 7 after injury. Late phase (Day 24) of wound repair was characterized by a decrease in mRNA and protein expression and a decrease in gelatinolytic activity of MMP-2 in all post-injured samples. On the contrary, high (p < 0.001) levels of mRNA expression, prominent pro-and active forms of MMP-9 and cells immunopositive for MMP-9 were present exclusively in the post-injured tissues from nude mice on Day 24 after wounding. This data suggest that MMP-9 expression in the remodeling phase of wound healing in nude mice could be a major component of their ability for scar-free healing.     

Periostin localizes to cells in normal skin, but is associated with the extracellular matrix during wound repair

Epidermal tissue repair represents a complex series of temporal and dynamic events resulting in wound closure. Matricellular proteins, not normally expressed in quiescent adult tissues, play a pivotal role in wound repair and associated extracellular matrix remodeling by modulating the adhesion, migration, intracellular signaling, and gene expression of inflammatory cells, pericytes, fibroblasts and keratinocytes. Several matricellular proteins show temporal expression during dermal wound repair, but the expression pattern of the recently identified matricellular protein, periostin, has not yet been characterized. The primary aim of this study was to assess whether periostin protein is present in healthy human skin or in pathological remodeling (Nevus). The second aim was to determine if periostin is expressed during dermal wound repair. Using immunohistochemistry, periostin reactivity was detected in the keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin. In pathological nevus samples, periostin was present in the extracellular matrix. In excisional wounds in mice, periostin protein was first detected in the granulation tissue at day 3, with levels peaking at day 7. Periostin protein co-localized with α-smooth muscle actin-positive cells and keratinocytes, but not CD68 positive inflammatory cells. We conclude that periostin is normally expressed at the cellular level in human and murine skin, but additionally becomes extracellular during tissue remodeling. Periostin may represent a new therapeutic target for modulating the wound repair process.     

Spatiotemporal expression of periostin during skin development and incisional wound healing: lessons for human fibrotic scar formation

Differentiation of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from embryonic through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the embryonic deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming growth factor beta 1 inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.     

Actin-dependent deposition of putative endosomes and endoplasmic reticulum during early stages of wound healing in characean internodal cells

We investigated the behaviour of organelles stained with FM1-43 (putative endosomes) and/or LysoTracker Red (LTred; acidic compartments) and of the endoplasmic reticulum (ER) during healing of puncture and UV-induced wounds in internodal cells of Nitella flexilis and Chara corallina. Immediately after puncture, wounds were passively sealed with a plug of solid vacuolar inclusions, onto which a bipartite wound wall was actively deposited. The outer, callose-containing amorphous layer consisted of remnants of FM1-43- and LTred-labelled organelles, ER cisternae and polysaccharide-containing secretory vesicles, which became deposited in the absence of membrane retrieval (compound exocytosis). During formation of the inner cellulosic layer, exocytosis of secretory vesicles with the newly formed plasma membrane is coupled to endocytosis via coated vesicles. Migration of FM1-43- and LTred-stained organelles, ER and secretory vesicles towards the cell cortex and deposition of a bipartite wound wall could also be induced by spot-like irradiation with ultraviolet light. Cytochalasin D reversibly inhibited the accumulation and deposition of organelles. Our study indicates that active actin-dependent deposition of putative recycling endosomes is required for wound healing (plasma membrane repair) and supports the hypothesis that deposition of ER cisternae helps to restore wounding-disturbed Ca(2+) metabolism.     

Actin microdomains on endothelial cells: association with CD44, ERM proteins,and signaling molecules during quiescence and wound healing

During studies of the actin cytoskeleton in cultured endothelial cells we have observed that the luminal side of many cells contains F-actin microdomains that are rich in the hyaluronan receptor CD44 and in ezrin-radixin-moesin (ERM) proteins. A small subpopulation of the domains are also enriched in tyrosine phosphorylated proteins and signaling molecules. Confocal microscopy of rat aortic endothelial cells in situ demonstrated that similar microdomains occur in vivo. During healing of endothelial wounds, characteristic alterations of the actin cytoskeleton occurred. Thus, in many cells close to the wound, focal F-actin branching points appeared. The branching points were similar to the microdomains in that they colocalized with CD44 and ERM proteins, but, in addition, they formed centers for actin filament branching and were associated with phosphorylated protein kinase C /_II. These colocalization data are consonant with the view that activated PKC is responsible for activating ERM-mediated crosslinking between CD44 and the actin cytoskeleton. Importantly, inhibition of PKC activity decreased staining for phosphorylated ERM proteins, decreased the frequency of F-actin branching points, and inhibited monolayer wound healing. Together, our data show that endothelial cells contain a novel actin cytoskeletal structure, the F-actin microdomain, and suggest that during wound healing such structures become associated with activated signaling molecules and thereby enhance actin cytoskeletal remodeling.