Proteomic profiling identifies an UV-induced activation of cofilin-1 and destrin in human epidermis.

The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin.

Endothelial network formed with human dermal microvascular endothelial cells in autologous multicellular skin substitutes.

A human skin equivalent from a single skin biopsy harboring keratinocytes and melanocytes in the epidermal compartment, and fibroblasts and microvascular dermal endothelial cells in the dermal compartment was developed. The results of the study revealed that the nature of the extracellular matrix of the dermal compartments plays an important role in establishment of endothelial network in vitro. With rat-tail type I collagen matrices only lateral but not vertical expansion of endothelial networks was observed. In contrast, the presence of extracellular matrix of entirely human origin facilitated proper spatial organization of the endothelial network. Namely, when human dermal fibroblasts and microvascular endothelial cells were seeded on the bottom of an inert filter and subsequently epidermal cells were seeded on top of it, fibroblasts produced extracellular matrix throughout which numerous branched tubes were spreading three-dimensionally. Fibroblasts also facilitated the formation of basement membrane at the epidermal/matrix interface. Under all culture conditions, fully differentiated epidermis was formed with numerous melanocytes present in the basal epidermal cell layer. The results of the competitive RT-PCR revealed that both keratinocytes and fibroblasts expressed VEGF-A, -B, -C, aFGF and bFGF mRNA, whereas fibroblasts also expressed VEGF-D mRNA. At protein level, keratinocytes produced 10 times higher amounts of VEGF-A than fibroblasts did. The generation of multicellular skin equivalent from a single human skin biopsy will stimulate further developments for its application in the treatment of full-thickness skin defects. The potential development of biodegradable, biocompatible material suitable for these purposes is a great challenge for future research.

Fibroblasts facilitate re-epithelialization in wounded human skin equivalents.

The re-epithelialization of the wound involves the migration of keratinocytes from the edges of the wound. During this process, keratinocyte migration and proliferation will depend on the interaction of keratinocytes with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal-epidermal junction (DEJ) and (2) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of keratinocyte growth factor and especially by epidermal growth factor. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for keratinocyte migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.

Recessive epidermolysis bullosa simplex phenotype reproduced in vitro: ablation of keratin 14 is partially compensated by keratin 17.

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitro the bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14-/-) and dyskeratotic (K14-/-) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitro but not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14-/- keratinocytes in vitro.

Induction of HSP27 nuclear immunoreactivity during stress is modulated by vitamin C.

For the investigation of the skin irritancy potential of chemicals in an in vitro model it is necessary to have sensitive endpoints that predict the effects of those compounds on native human skin. Recently, we have identified that 27-kDa heat shock protein (HSP27) can serve as a sensitive marker of skin irritation, as exposure of human skin to sodium lauryl sulfate (SLS) both in vitro and in vivo induced relocalization of HSP27 from the cytoplasm to the cell nucleus. The aim of the present study was to determine whether nuclear localization of HSP27 could be used as a parameter for evaluation of potential skin irritants in screening assays in vitro. For this purpose, human skin equivalent consisting of epidermis reconstructed on de-epidermized dermis was exposed to SLS or UV light. Stress-induced nuclear relocalization of HSP27 was observed in excised skin exposed to SLS or UV light and in reconstructed epidermis only when the latter was generated in the absence of vitamin C. The omission of vitamin C results in an impaired barrier function. In the presence of vitamin C, however, the barrier function was comparable with excised skin, suggesting that vitamin C may control the response to stress in the reconstructed epidermis. Besides the presence of vitamin C, the response of skin equivalents may strongly depend on other conditions under which they are generated, because the stress-induced HSP27 relocalization was not detected in the commercially available epidermal kit EpiDerm. The results of the present study show that HSP27 nuclear staining can serve as a sensitive marker for skin irritation or cellular stress in excised skin as well as in certain well-characterized human skin equivalents in vitro.