Mutsumi Okazaki

The mechanism of epidermal hyperpigmentation in café-au-lait macules of neurofibromatosis type 1 (von Recklinghausen's disease) may be associated with dermal fibroblast-derived stem cell factor and hepatocyte growth factor

Summary Backgroundu2003The mechanism of the accentuated melanization in café‐au‐lait macules (CALMs) in patients with neurofibromatosis type 1 (NF1; von Recklinghausens disease) has not been elucidated.

Effects of all-trans retinoic acid on melanogenesis in pigmented skin equivalents and monolayer culture of melanocytes

The effects of all-trans retinoic acid (RA) on melanogenesis and the mechanism of its action in topical treatment have not been elucidated. The purpose of this study was to determine the effects of RA on melanogenesis in the pigmented skin equivalent as well as in monolayer culture of melanocytes, and to determine whether RA, hydroquinone (HQ), and hydrocortisone (HC) show synergistic depigmenting effects in combined treatments of each other. The suppressing effect of RA on melanogenesis was not observed in pigmented skin equivalents and monolayer culture of murine and human melanocytes, although HQ showed strong inhibition of melanogenesis. The synergistic effects between RA, HQ, and HC were not particularly seen. The results suggested that RA neither has direct inhibitory effects on melanogenesis of melanocytes, nor influences the cell-cell interactions between melanocytes, keratinocytes and fibroblasts, such as paracrine actions with regard to melanin production. The role of RA in bleaching treatments appears to be in other specific actions, such as promotion of keratinocytes proliferation and acceleration of epidermal turnover.

Choice of osseous and osteocutaneous flaps for mandibular reconstruction.

Microvascular free flap transfer currently represents one of the most popular methods for mandibularreconstruction. With the various free flap options nowavailable, there is a general consensus that no single kindof osseous or osteocutaneous flap can resolve the entire spectrum of mandibular defects. A suitable flap, therefore, should be selected according to the specific type of bone and soft tissue defect. We have developed an algorithm for mandibular reconstruction, in which the bony defect is termed as either “lateral” or “anterior” and the soft-tissue defect is classified as “none,” “skin or mucosal,” or “through-and-through.” For proper flap selection, the bony defect condition should be considered first, followed by the soft-tissue defect condition. When the bony defect is “lateral” and the soft tissue is not defective, the ilium is the best choice. When the bony defect is “lateral” and a small “skin or mucosal” soft-tissue defect is present, the fibula represents the optimal choice. When the bony defect is “lateral” and an extensive “skin or mucosal” or “through-and-through” soft-tissue defect exists, the scapula should be selected. When the bony defect is “anterior,” the fibula should always be selected. However, when an “anterior” bone defect also displays an “extensive” or “through-and-through” soft-tissue defect, the fibula should be usedwith other soft-tissue flaps. Flaps such as a forearm flap, anterior thigh flap, or rectus abdominis musculocutaneous flap are suitable, depending on the size of the soft-tissue defect.

Tretinoin reverses upregulation of matrix metalloproteinase-13 in human keloid-derived fibroblasts

Abstract Keloids are skin abnormalities that are characterized by excessive deposition of collagen bundles in the dermis. Patients with keloids complain not only about their cosmetic appearance, but also about continuous itching and/or tenderness associated with chronic inflammation. Degradation of extracellular matrix (ECM) may be upregulated, associated with the expansion of keloids into circumferential skin, and high metabolic activity of keloid tissues may be due to increased matrix metalloproteinase (MMP) activity. Based on these hypotheses, we examined differences in expression of MMP‐1, MMP‐8, and MMP‐13 between keloid‐derived and normal dermal fibroblasts. Since retinoids are potent inhibitors of MMPs in the treatment of photoaged skin and cancers, we also examined whether or not tretinoin affects MMP expression of keloid‐derived fibroblasts. The results of real‐time polymerase chain reaction and ELISA demonstrated significant upregulation of MMP‐13 and significant downregulation of MMP‐1 and MMP‐8 in keloid‐derived fibroblasts, at both mRNA and protein levels. MMP‐1 mRNA expression in the control group was significantly upregulated after the addition of tretinoin, whereas no significant change was observed in the keloid group. MMP‐8 mRNA expression in the control group was significantly upregulated by tretinoin, with the peak at 12u2003h, while no significant change was observed in the keloid‐derived fibroblasts. In contrast, the remarkably elevated MMP‐13 mRNA expression in the keloid group was significantly suppressed, with the peak suppression at 12u2003h after addition of tretinoin, while MMP‐13 mRNA expression in the control group was not significantly changed. The decrease in MMP‐1 and MMP‐8 may contribute to accumulation of type I and type III collagen in keloid tissues, and this mechanism may be modulated by molecular interaction with MMP‐13. Tretinoin appeared to reverse the abnormal expression profile of MMPs in keloid‐derived fibroblasts, such as markedly elevated expression of MMP‐13, partly through inactivation of AP‐1 pathway. The present results suggest that tretinoin may be clinically useful to improve the chronic inflammation seen in keloids and prevent expansion of keloid tissues into circumferential normal skin.

Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts

Backgroundu2002 The majority of skin changes associated with ageing are caused by photoageing and reflect cumulative sun exposure. Although the actinic damage plays a major role in skin pigmentation, it is also important to examine the effects of chronological cellular ageing on the pigmentation. The chief cellular components of the skin other than melanocytes are keratinocytes and fibroblasts, and the influences of age‐related changes in those cells on skin pigmentation have not been elucidated.

Hepatic Artery Reconstruction with Double- Needle Microsuture in Living-Donor Liver Transplantation

In living‐donor liver transplantation (LDLT), reconstruction of the hepatic artery is challenging because the recipient artery is located deep in the abdominal cavity and the operating field is limited. Also, the hepatic artery of the graft is short and the recipient artery is occasionally damaged. To overcome these difficulties, we developed a double‐needle microsuture technique for artery reconstruction. A total of 161 adult patients received 163 LDLTs using this new technique. The first suture was placed at the most difficult point in the artery to be visualized through the microscope. Each stitch was placed from the inner side of the arterial wall to the outer side. The posterior stitch was tied pulling toward the back. The subsequent sutures were advanced anteriorly on either side adjacent to the previous suture. Hepatic artery thrombosis occurred in 4 patients (2.5%), only 2 (1.2%) of which were associated with arterial reconstruction. Intimal dissection developed in the recipient artery in 2 patients (1.2%). Three (50%) of these 6 complications occurred more than 10 days after LDLT. In conclusion, this suturing technique allows for safe intimal adaptation even when the arterial tunica intima is separated from the tunica media, because all stitches are carried from inside of the vessel to the outside, contributing to more satisfactory results. Liver Transpl 12:46–50, 2006.

Elevated expression of hepatocyte and keratinocyte growth factor in cultured buccal-mucosa-derived fibroblasts compared with normal-skin-derived fibroblasts

Oral mucosa heals faster with less scar formation than skin and a hypertrophic scar is very rare in the oral cavity, but its mechanism has not been elucidated enough. To elucidate whether or not there are differences in growth factor expression between fibroblasts derived from buccal mucosal and normal skin, we investigated the expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) by cultured fibroblasts. The semiquantitative RT-PCR revealed that the expression of HGF and KGF transcripts by buccal mucosal fibroblasts was significantly elevated compared with that by dermal fibroblasts. In parallel, ELISA revealed the significant increase of HGF production by buccal mucosal fibroblasts. The level of production of SCF protein did not differ significantly. Our study suggests that increased expression of HGF and KGF by buccal mucosal fibroblasts may partly be responsible for the faster wound healing with less scar formation in the oral cavity compared with normal skin.

Differential expression of heparin-binding EGF-like growth factor (HB-EGF) mRNA in normal human keratinocytes induced by a variety of natural and synthetic retinoids

Abstract It was recently revealed that epidermal growth following topical treatment with all‐trans retinoic acid (atRA) was at least partly induced by heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) released from suprabasal keratinocytes. Since proliferation of keratinocytes appears to be one of the critical roles of atRA in depigmentation treatment and promotion of wound healing, HB‐EGF is considered suitable for assessing the therapeutic value of topical retinoids. In this study, HB‐EGF mRNA expression in normal human keratinocytes after atRA treatment was examined, and the effects of a variety of natural and synthetic retinoids were compared. The results of reverse transcription polymerase chain reaction (RT‐PCR) suggested that induction of differentiation increased HB‐EGF mRNA expression in cultured keratinocytes. Real‐time PCR analyses revealed that HB‐EGF mRNA expression was elevated dose‐dependently with atRA, peaking at 12u2003h. This elevation was more prominent in confluent keratinocytes than in subconfluent cells, suggesting that differentiated keratinocytes are more subject to stimulation of HB‐EGF expression by atRA than proliferating keratinocytes. HB‐EGF mRNA was upregulated in differentiation‐induced keratinocytes by all retinoids used in this study at 1u2003µmol/l, and marked upregulation was seen when treated with three isotypes of retinoic acid (atRA, and 9‐cis and 13‐cis retinoic acid). RARα‐selective agonists (Am80, Am580, ER‐38925, and TAC‐101) and a panagonist of RARs (Re80) caused relatively low elevation of HB‐EGF transcripts, as did all‐trans retinol (Rol) and all‐trans retinal (Ral). Although another panagonist (Ch55) showed the highest elevation of HB‐EGF mRNA, it was relatively cytotoxic at the concentration employed. Ral and Rol were found to upregulate HB‐EGF when used at 100u2003µmol/l to 1u2003mmol/l, to a similar extent of atRA at 1–10u2003µmol/l. The capacity of retinoids to upregulate HB‐EGF may be an important index for investigation and development of an ideal synthetic retinoid, which has maximum benefits and minimum side‐effects

Construction of pigmented skin equivalent and its application to the study of congenital disorders of pigmentation

We have constructed a pigmented skin equivalent and used it to study the hyperpigmentation seen in café-au-lait macules to elucidate whether the pigmented skin equivalent could be used as a model of congenital hyperpigmentary disorders. When we used fibroblasts derived from café-au-lait macules of neurofibromatosis type 1, the amount of pigment was significantly greater than in models using cells derived from normal skin. Quantities of pigment were not seen when keratinocytes derived from solitary café-au-lait macules were used, a possible reason being that keratinocytes on the skin equivalent are in a proliferating condition and are not well-differentiated enough to act on other cells. Our results suggested that our pigmented skin equivalent is useful for the study of congenital hyperpigmentary disorders, although insufficient differentiation of keratinocytes might be a disadvantage.

Epidermal hyperpigmentation in non-syndromic solitary cafe-au-lait macules may be associated with increased secretion of endothelin-1 by lesional keratinocytes.

To clarify the mechanism of accentuated melanisation in non-syndromic solitary cafe-au-lait macules we used an enzyme-linked immunosorbent assay (ELISA) to measure the concentration of melanogenic cytokines secreted by cultured keratinocytes and fibroblasts derived from the skins of the macules and compared them with those derived from normal people. Endothelin-1 (ET-1) was significantly increased in cultured keratinocytes in the macules compared with the normals. In contrast, the secretion of other cytokines secreted by keratinocytes or fibroblasts did not differ between the groups. It may be that the increased secretion of ET-1 by epidermal keratinocytes has a role in the accentuated epidermal melanisation seen in non-syndromic macules.