The plant alkaloid conophylline inhibits matrix formation of fibroblasts.

Conophylline is a Vinca alkaloid from leaves of the tropical plant Ervatamia microphylla and has been shown to mimic the effect of the growth and differentiation factor activin A on pancreatic progenitor cells. However, activin A stimulates fibrosis of pancreatic stellate cells, whereas conophylline inhibits it, suggesting that this compound may serve as an antifibrotic drug. Here we investigated the effects of conophylline on human foreskin fibroblasts, especially focusing on extracellular matrix (ECM) proteins. A gene microarray analysis revealed that conophylline remarkably suppressed expression of the gene for hyaluronan synthase 2 (HAS2) and of its antisense RNA, whereas the expression of collagen genes was unaffected. Of note, immunostaining experiments revealed that conophylline substantially inhibits incorporation of versican and collagens into the ECM in cells treated with transforming growth factor ß (TGFß), which promotes collagen synthesis, but not in cells not treated with TGFß. Moreover, a protein biosynthesis assay disclosed that conophylline decreases collagen biosynthesis, concomitant with a decrease in total protein biosynthesis, indicating that conophylline-mediated inhibition of fibrosis is not specific to collagen synthesis. Conophylline affected neither TGFß-induced nuclear translocation of SMAD family member 2/3 (SMAD2/3) nor phosphorylation of SMAD2. However, conophylline substantially inhibited phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), suggesting that conophylline inhibits HAS2 expression via TGFß-mediated activation of the ERK1/2 pathway. Taken together, our results indicate that conophylline may be a useful inhibitor of ECM formation in fibrosis.

Minimally invasive endoscopic osteosynthesis for frontozygomatic fracture: a new approach.

Miniplate fixation on the lateral face of the orbital rim using existing endoscopic methods for frontozygomatic fracture still has some disadvantages, such as cosmetic disturbance from the lateral brow incision for the trocar, and abnormal palpability of the miniplate. We applied a new method of endoscopic osteosynthesis by access through temporal incisions alone and miniplate fixation on the lateral temporal face of the frontozygoma. Postoperative courses were uneventful in all four cases treated, and we achieved cosmetic improvement (minimizing incision and scars) as well as decreased palpability of the miniplate.

Versican contributes to ligament formation of knee joints.

Versican is a large proteoglycan in the extracellular matrix. During embryonic stages, it plays a crucial role in the development of cartilage, heart, and dermis. Previously, we reported that Prx1-Vcan conditional knockout mice, lacking Vcan expression in mesenchymal condensation areas of the limb bud, show the impaired joint formation and delayed cartilage development. Here, we investigated their phenotype in adults and found that they develop swelling of the knee joint. Histologically, their newborn joint exhibited impaired formation of both anterior and posterior cruciate ligaments. Immunostaining revealed a decrease in scleraxis-positive cells in both articular cartilage and ligament of Prx1-Vcan knee joint, spotty patterns of type I collagen, and the presence of type II collagen concomitant with the absence of versican expression. These results suggest that versican expression during the perinatal period is required for cruciate ligaments' formation and that its depletion affects joint function in later ages.

Effects of Fibroblast Growth Factor 2 on Burn Injury and Repair Process: Analysis Using a Refined Mouse Model.

BACKGROUND: Burn injury is one of the most debilitating traumas, which induces multiple organ dysfunctions, resulting in high levels of morbidity and mortality. Fibroblast growth factor 2 (FGF2) has been applied to burn injury, whose precise mechanisms underlying facilitating the healing have not been fully understood. Although various animal models have been developed in pigs, rabbits, rats, and mice, no mouse model that creates burns consistent in their extent and depth have not been developed. Here, we developed a mouse burn model, and investigated details of the burn process, and elucidated the mechanisms of FGF2 effects. METHODS: A device with an 8-mm metal probe and a temperature controller was developed, which controls the temperature of the probe. Using the device, 1 or 2 of full-thickness burn injuries were generated on the back under catagen/telogen of 6-month-old C57BL/6 male mice. After 24 hours, FGF2 or phosphate-buffered saline was injected into the injured region, and at days 3, 5, and 7, histological and immunohistochemical analysis was performed to observe the injury and repair process. RESULTS: The device constantly generated a mouse full-thickness burn injury. The repair was initiated on the bottom of the burn as well as the margin. Local treatment with FGF2 displayed higher levels of immunostaining for both CD31+ and alpha-smooth muscle actin. CONCLUSIONS: The device we developed is useful to generate a mouse burn injury model. FGF2 facilitates tissue repair with an increased number of both CD31+ and αSMA+ cells.

5-Aminolevulinic acid-based photodynamic therapy for the treatment of two patients with extramammary Paget's disease.

Photodynamic therapy (PDT), which employs a combination of a tumor-localizing photosensitizer and visible light, has been used in the treatment of extramammary Paget's disease (EMPD). Two patients with EMPD were treated with PDT using 5-aminolevulinic acid (ALA). Histologically, in both cases, Paget's cells were present within the epidermis. Case 1 was a 92-year-old male who underwent total extirpation for treatment of EMPD. Two topical ALA-PDT treatments were applied to parts of the lesions at a total dose of 200J/cm2. Case 2 was a 73-year-old female, whose lesions in the right labia majora were treated with 3 topical ALA-PDT sessions at a total dose of 300 J/cm2. Clinical findings after the irradiation showed improvement in both patients, and elimination of tumor cells in the epidermis was confirmed histologically. Case 1 had no recurrence in the irradiation field at three months after PDT. Case 2 had a recurrence only in the periphery parts of the lesions at two months after PDT, but the periphery lesions remitted with two more PDT treatments. Topical ALA-PDT is an effective treatment for EMPD with tumor cells within the epidermis. It is noninvasive and achieves a cosmetically excellent outcome, especially in elderly patients and those in poor general condition.

Lymphoepithelioma-like carcinoma of the skin with potential for sweat glandular differentiation.

Lymphoepithelioma-like carcinoma of the skin (LELCS) is a cutaneous malignancy with histopathological resemblance to lymphoepithelioma of the nasopharynx. Its histogenesis remains unknown, and few cases showing skin appendage differentiation have been reported to date. We present the case of a 77-year-old Japanese male with an asymptomatic red nodule on his left cheek. Because the histopathological study revealed focal growth of tumor cells lacking connections with the epidermis and marked lymphocytic infiltration surrounding the neoplastic cell nests, the case was diagnosed as LELCS. On immunohistological staining, the neoplastic cells were positive for carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), multi-cytokeratin (CK), CK6, CK18, and CK19. On the basis of these results, we suggested that skin appendage differentiation, particularly sweat glandular differentiation, was present in this case.

High-resolution 400K oligonucleotide array comparative genomic hybridization analysis of neurofibromatosis type 1-associated cutaneous neurofibromas.

Neurofibromatosis type 1 (NF1) is a genetic disorder where affected individuals develop benign or malignant nervous system tumors. To date, NF1 is caused by mutations in the NF1 tumor suppressor gene located at chromosome band 17q11.2. In this study, we aimed to characterize novel recurrent regional chromosomal imbalances and tumor-related candidate genes in NF1-associated cutaneous neurofibromas. Nine cutaneous neurofibromas from NF1 patients were screened for recurrent chromosomal imbalances using high-resolution 400K oligonucleotide array comparative genomic hybridization (aCGH). All the cases exhibited at least one sub-microscopic abnormality. Regions of recurrent chromosomal imbalances in a least one third of cases were loss of 1q13.2 (33%, FAM19A3), 1q21.1 (44%, RABGAP1L), 2q37.1 (56%, INPP5D), 3p25.1 (67%, CHCHD4), 4p15.32 (56%, FGFBP1), 5q11.2 (56%, ARL15), 6q22.31 (56%, NKAIN2), 6q22.33 (67%, ARHGAP18), 6q25.1 (67%, UST), 7q13 (56%, ADCY1), 12q13.13 (44%, KRT71), 19q13.32 (56%, GRLF1), and 20p11.21 (56%, NLP) and gain of 2p23.3 (76%, C2orf53), 8q22.3 (44%, ODF1) and 8q24.3 (67%, ARC). Several chromosomal imbalances, including loss of 7q11.23, 13q14.1, 14q32.13, 17p12, and 17q11.2 were detected at a lower frequency. We also confirmed that these chromosomal imbalances were not detected in the patient-matched lymphocyte DNAs. Amongst the 6 tumor-related candidate genes (RABGAP1L, ADCY1, SLIT2, GRLF1, UST, and ARC) identified in the regions of recurrent chromosomal imbalances, the gene expression changes of UST (down-regulation) and ARC (up-regulation) were found to be significantly associated with copy number alterations. The novel recurrent chromosomal imbalances and the altered expression levels of the tumor-related candidate genes may be associated with the development of NF1-associated benign cutaneous neurofibromas.

YAG laser treatment causes rapid degeneration and regeneration of collagen fibres in pig skin and facilitates fibroblast growth.

The non-ablative laser therapies have been speculated to cause microinjury in the dermal collagen fibres and increase collagen synthesis in the fibroblasts, leading to remodelling of the extracellular matrix. This study investigated the effects of neodymium YAG laser treatment on pig skin, especially focusing on its extracellular matrix molecules. The dorsal areas of a minipig were subjected to laser treatment, and samples were obtained by punch biopsies, and histological, immunohistochemical, and biochemical analyses were performed. The laser treatment caused degeneration of collagen fibres and fibrils, which were reconstituted within 24 hours, whereas there was no inflammation and no apparent damage on elastic fibres. Small blood vessels disappeared by the laser treatment, which re-appeared in 3 days. Biochemically, the amounts of collagen decreased up to day 3 after the treatment and then increased at day 7. When fibroblasts in dermal tissue at day 28 were counted, more fibroblasts in the treated tissue were observed than non-treated control. These results suggest that, although the laser treatment transiently degenerates collagen fibres and fibrils, it restores and increases them, mainly by an increase in dermal fibroblasts, assuring its minimal complication of skin.

Lapachol suppresses cell proliferation and secretion of interleukin-6 and plasminogen activator inhibitor-1 of fibroblasts derived from hypertrophic scars.

OBJECTIVES: The pathogenesis and therapy of hypertrophic scar have not yet been established. Our aim was to investigate the antiproliferative and antisecretory effects of lapachol, isolated from the stem bark of Avicennia rumphiana Hall. f., on hypertrophic scar fibroblasts. METHODS: The effects of lapachol on hypertrophic scar fibroblast proliferation were measured using the MTT assay, cell-cycle analyses and lactate dehydrogenase assays. The type I collagen α-chain (COL1A1), interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) mRNA and/or protein levels of hypertrophic scar-fibroblasts were quantitated by real-time PCR and ELISA. KEY FINDINGS: Lapachol at 25 and 50 µm significantly inhibited the in vitro proliferation of hypertrophic scar fibroblasts, but not fibroblasts from non-lesional skin sites. In addition, lapachol had no apparent effect on cell cycle and lactate dehydrogenase activity in conditioned medium from lapachol-treated hypertrophic scar fibroblasts was nearly equal to that in medium from vehicle-treated cells. Lapachol treatment also inhibited COL1A1 and PAI-1 mRNA levels in hypertrophic scar fibroblasts, but did not affect IL-6 mRNA levels. The protein levels of IL-6 and PAI-1 in conditioned medium from hypertrophic scar fibroblasts treated with 50 µm lapachol were lower than those from vehicle-treated hypertrophic scar fibroblasts. CONCLUSIONS: Lapachol decreased the proliferation rate of hypertrophic scar fibroblasts. As IL-6 and PAI-1 secretion was also lowered in lapachol-treated hypertrophic scar fibroblasts, our findings suggested that lapachol may have suppressed extracellular matrix hyperplasia in wound healing and possibly alleviated the formation of hypertrophic scar.