Kuniko Kadoya

The importance of laminin 5 in the dermal–epidermal basement membrane

The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal-dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal-dermal junction is essential to stability of the skin.

Protective effect of matrix metalloproteinase inhibitors against epidermal basement membrane damage: skin equivalents partially mimic photoageing process.

Background  The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis, and in controlling epidermal differentiation. The BM has been reported to be damaged in sun‐exposed skin. Although matrix metalloproteinases (MMPs) are believed to be involved in the BM damage, there is no good in vitro model for examining BM damage by MMPs or for exploring methods to protect the BM.

Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis

Background  Fibulin‐5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin‐5 in human skin and its changes during the ageing process are not known.

Evaluation of autologous cultured epithelium as replacement skin after tattoo excision: correlation between skin texture and histological features.

Summary  Background Cultured epidermal autographs (CEAs) are currently used as a coverage treatment for burn wounds, for disfiguring burn scars involving depigmentation and in restoring the elasticity of the skin. The advantage of CEAs is that epidermal sheets prepared from small skin pieces can be enlarged sufficiently to cover large burn areas.

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six‐grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Secretion of GAWK, a Presumed Product of Chromogranin B Processing, by a Human Medullary Thyroid Carcinoma Cell Line: Effect of Phorbol Ester, Forskolin and Calcium-lonophore.

GAWK was isolated from human pituitary glands and presumed to be a processing product of human Chromogranin B. In the present studies, we investigated effects of phorbol ester (TPA), forskolin and Ca2+‐ionophore (A23187) on GAWK secretion using a human medullary thyroid carcinoma‐derived cell line (TT‐cell). A dose‐responsive stimulation of GAWK‐like immunoreactivity (LI) secretion was found in the presence of TPA as well as forskolin. A23187 caused an increase of GAWK‐LI secretion and addition of TPA together with A23187 revealed an additive effect on the GAWK‐LI secretion. These findings indicate that GAWK‐LI is a secretory peptide in the TT‐cells and suggest that A and C kinase, and possibly Ca2+‐calmodulin transduction systems are involved in the GAWK‐LI secretion by the TT‐cells. TT‐cells may provide a good model for the investigation of regulation of Chromogranin secretion.

Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin‐1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin‐1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.

Discordant secretion of calcitonin and chromogranin in the human medullary thyroid carcinoma cell line

A cell fine of human medullary carcinoma of the thyroid, abundantly producing calcitonin (Ct) and related hormones, has proved remarkably useful as an endocrine tumor model for the study of the secretion mechanism. This cell line (TT cell) was used in studies to elucidate the dynamics of the release of Ct and chromogranin (Cg) to culture medium. The studies evaluated the intracellular concentration of Ct and Cg and the concentration changes elicited by the protein kinase C activator, phorbol ester (TPA); the adenylate cyclase-associated protein kinase A activator, forskolin; and the calcium ionophore, A23187. In addition, immunogold labeling of Ct and Cg was carried out to investigate the ultrastructural changes resulting from the stimulations. All these secretagogues effected the release of Ct and Cg into the medium in a dose-dependent manner, and the rate of the increase in the Ct secretion was consistently and markedly higher than that of Cg in more than certain dosages of the secretagogues. Most cells contained secretory granules immunolabeled for both Ct and Cg, and a considerable decrease was noted in the poststimulation count of the granules containing both substances, with the cells retaining more Cg than Ct. The discordance may be explained by different secretory pathways of the two proteins or different rates of synthesis.

Pretreatment of human keratinocyte sheets with laminin 5 improves their grafting efficiency

Laminin 5 is essential in epithelial attachment to stromal tissues, suggesting that it might improve keratinocyte attachment in a variety of clinical situations. In this study, we examined the effect of exogenous laminin 5 upon the efficiency of transplantation of keratinocyte sheets in animal models. Keratinocyte sheets were prepared according to the method of Rheinwald & Green (1975). Purified laminin 5 was added to the sheets of group 1 (1.0 microg per cm2), Dulbeccos modified Eagles medium alone was added to group 2. The sheets were grafted to the panniculus carnosus of nude mice (BALB/C nu/nu) (n = 12) and nude rats (Fisher 344) (n = 15). The take rate was assessed by measurement of the area of surviving epithelium at 7 d postgrafting. Laminin 5 bound the keratinocyte sheets of group 1. At 7 d postgrafting, the area of epithelialization of group 1 was significantly larger than that of group 2. Immunohistochemistry staining showed that collagen IV, laminin 5, and collagen VII stained more strongly at the dermal-epidermal junction in group 1 than in group 2. Integrin chains alpha6 and beta4 were similar in both groups. Electron microscopy at day 3 after grafting, showed the lamina densa of group 1 to be more continuous than in group 2. Pretreatment of cultured human keratinocyte sheets with laminin 5 improved the extent of epithelial coverage and increased the rate of neobasement membrane formation. The results suggest that laminin 5 promotes epithelial attachment by increasing the rate of basement membrane assembly.