Masaharu Ichinose

Revascularization determines volume retention and gene expression by fat grafts in mice.

Autologous fat transplantation is a popular and useful technique in plastic and reconstructive surgery. The efficiency and survival of such grafts is predictable in many cases, but there are still issues to be resolved, such as how to improve graft volume retention. To address the issue of volume retention, we studied the effect of revascularization from the recipient on the size and function of adipocytes in fat grafts. Treatment of mice with TNP-470, an angiogenesis inhibitor, reduced blood flow from the recipient into the graft after subcutaneous transplantation of epididymal fat. The weight of transplanted tissues and the size of adipocytes in the grafts were significantly lower in mice treated with TNP-470 (TNP mice) than in control mice. Expression of genes for enzymes related to lipid accumulation was decreased in the grafts of TNP mice compared with control mice. Moreover, the expression of adipocyte-derived angiogenic peptides, VEGF and leptin, was significantly lower in the grafts of TNP mice than in grafts from control animals. The expression of VEGF and leptin by cultured human adipocytes was increased in the presence of conditioned medium from cultured vascular endothelial cells. These results show that the inhibition of the revascularization of fat grafts after transplantation reduces graft volume retention and cellular function. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft.

CD151 dynamics in carcinoma-stroma interaction: integrin expression, adhesion strength and proteolytic activity.

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and α3β1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5CD151−), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5CD151−, striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. α3β1 integrin was internalized in part, and α6β4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5CD151−. Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma–stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.

A new method for the treatment of graft infection in the thoracic aorta: in situ preservation

BACKGROUND We have developed a new method to control graft infection by a combination of two procedures, extensive disinfection followed by tissue flap implantation, allowing preservation of the original graft. METHOD Soon after the diagnosis of graft infection was confirmed, the wound was re-explored, and debridement, irrigation and packing with sponges soaked with 10% iodine solution were employed. This procedure was repeated every 8 hours for the first 48 hours. For the second step, tissue flaps using omentum or muscle were implanted around the graft as well as in dead space, and the wound was closed primarily. MATERIALS A total of 6 patients were treated: 4 in the acute and 2 in the chronic phase of infection. The original procedures were a Bentall procedure + arch replacement (1), ascending aorta replacement + arch (3) and replacement of the descending aorta (2). In descending aorta cases, an extended thoracoplasty was concomitantly added to eliminate dead space in the pleural cavity. RESULTS Graft infections were controlled in all 6 patients. One hospital death unrelated to infection was encountered. Five patients were discharged, but 1 died of a stent-graft complication. The follow-up period ranged from 4 months to 10 years. CONCLUSION Our method of extensive disinfection followed by tissue flap coverage of the graft proved to be highly effective in controlling the serious complication of graft infection associated with surgery of the thoracic aorta.

DNA Methylation Changes During Cleft Palate Formation Induced by Retinoic Acid in Mice

Objectives: The aim of this study was to analyze epigenetic (specifically, DNA methylation) participation in the mechanisms of cleft palate only induced by maternal exposure to all-trans retinoic acid in mice. Design: Cleft palate only was induced in fetuses by maternal exposure to all-trans retinoic acid. Their secondary palates were excised for analysis. Cytosine extension assay and restriction landmark genomic scanning were performed to analyze DNA methylation status. The expression levels of the DNA methyltransferases were examined by real-time reverse transcriptase–polymerase chain reaction. Results: Using cytosine extension assay, on gestation day 14.5, the status of DNA methylation within CpG islands and in global DNA was decreased significantly in all-trans retinoic acid–treated groups compared with the controls (p < .01 and p < .05). In the controls, the status within CpG islands on gestation day 14.5 was significantly increased compared with gestation days 13.5 and 18.5 (p < .01). Using real-time reverse transcriptase–polymerase chain reaction, there was no significant change in the expression of DNA methyltransferases, except on gestation day 18.5. Using restriction landmark genomic scanning on gestation day 18.5, five spots (0.49%) in the controls and one spot (0.1%) in all-trans retinoic acid–treated groups were specifically detected. Conclusions: These results indicate that changes in DNA methylation may play an important role in the manifestation of cleft palate only caused by environmental factors such as maternal exposure to all-trans retinoic acid.

Cyclical Cell Stretching of Skin-Derived Fibroblasts Downregulates Connective Tissue Growth Factor (CTGF) Production

Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

A simple reconstruction for congenital unilateral lower lip palsy.

Background: Congenital unilateral lower lip palsy or congenital hypoplasia of the depressor anguli oris muscle, also known as asymmetric crying facies, characterized by deformity of the lower lip, lacks aggressive surgical intervention methods. Although several dynamic and static reconstruction methods have been reported, textbooks introduce only passive surgical intervention, of weakening the unaffected side through techniques such as selective marginal mandibular neurectomy which, however, tends to produce lack of emotive movement. Therefore, a new surgical intervention for the reconstructive treatment of asymmetric crying facies is presented. Methods: A bidirectional (horizontal and vertical) fascia was grafted to restore the aesthetic appearance of the asymmetric lower lip. The horizontal fascial strip achieves restoration of the center of the lower lip to its proper position, whereas the vertical fascial strip achieves aesthetic symmetry of the lower lip at mouth opening. Each end of the vertical strip is anchored to the lower lip and the mandibular bone, respectively, thereby allowing simultaneous movement of the mandible and lower lip. Results: The reconstruction of unilateral lower lip palsy has been successfully performed on seven patients, with ages ranging from 2 years 9 months to 11 years 1 month, since June of 1996. Conclusions: The aim of this procedure is not to achieve complete dynamic reanimation. However, with regard to its simplicity and minimal invasiveness and the satisfaction of the patients, it is considered to be a well-balanced surgical intervention.

Repair of wide coup de sabre without cutaneous excision by means of pericranial-galeal padding flap.

A case of double linear scleroderma of the forehead (coup de sabre) is described. The histopathology of this rare lesion is now well known with a normal epidermis and a sclerotic dermis. The correction was done with an original two-stage procedure: the lesion with alopecia was first treated by excision-suture and a transfer of the involved subcutaneous tissue along the right inner canthus; 1 year later, by a hemicoronal incision, we transferred a galeal-pericranial flap beneath the wider forehead lesion. We think that the use of a filling flap to correct wide coup de sabre lesions without cutaneous excision can be a simple alternative to the classic treatment by complete excision and flap reconstruction. The subcutaneous fascial system of the scalp can provide a good donor site with minimal morbidity.

Treatment of Nevus Ota by Liquid Nitrogen Cryotherapy

Nevus Ota is a disfiguring facial dermal melanosis that is observed rarely in Caucasians but is common in Asians. Until recently, carbon dioxide snow cryotherapy had been used most frequently to treat this disorder, but the results were not always satisfactory and often left scarring after treatment. During the past 15 years, we have treated a total of 600 cases of dermal pigmentary facial disfiguration using CRYO-MINI, a liquid nitrogen cryogenic instrument with a removable disk-shaped copper tip. We have found this method to be simple and extremely effective in the treatment of nevus Ota and senile lentigines, and it is also useful for treatment of delayed nevus spilus and blue nevus. This report presents our techniques and experiences as well as the results achieved with four patients who have a deeply situated type of nevus Ota. Associated problems to be considered in future treatment are discussed also.

Establishment of gene-vaccinated skin grafting against Toxoplasma gondii infection in mice.

Vaccine effects of in vivo gene-vaccinated skin graft were evaluated against Toxoplasma gondii (T. gondii) infection. By using a gene gun, cDNA coding T. gondii SAG1 molecule was intracutaneously vaccinated into C57BL/6 (B6; a susceptible strain), BALB/c (a resistant strain) and (C57BL/6 x BALB/c) F1 (CBF1) mice, and the gene-vaccinated skin of these strains was transplanted to CBF1 mice. Regarding the antibody production against SAG1, CBF1-recipient mice transplanted with the SAG1 gene-vaccinated B6 skin were high responders, whereas CBF1 mice skin grafted with vaccinated skin of both BALB/c and CBF1 mice were low responders. The donor-derived LC/DC migrated to the draining lymph nodes of the recipients from the skin graft within 3 days. The vaccine effect against T. gondii challenge infection was obtained in CBF1 mice which received the skin graft of the SAG1 gene-vaccinated BALB/c mice.

Tessier number 7 cleft with oblique clefts of bilateral soft palates and rare symmetric structure of zygomatic arch

Craniofacial clefts are very rare congenital deformities. Tessiers numbering classification is a simple system universally accepted. Excluding cleft lip and palate, the transverse or lateral cleft (Tessier number 7) is the most common type of craniofacial cleft, manifesting macrostomia without skeletal abnormalities. We present a case of Tessier number 7 cleft with oblique clefting of the soft palate bilaterally and rare symmetric structures of the zygomatic arch.