Toshikatsu Igata

Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1/2 and JNK pathways.

Background  Basic fibroblast growth factor (bFGF, FGF‐2) has been described as a multipotent cytokine that regulates cell growth as well as differentiation, matrix composition, chemotaxis, cell adhesion and migration in numerous cell types. It is known that bFGF stimulates proliferation of cultured fibroblasts. However, the detailed mechanism of fibroblast proliferation induced by bFGF in vitro still remains to be elucidated.

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type i collagen expression in scleroderma fibroblasts

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.

Hair miR-29a levels are decreased in patients with scleroderma

In the present study, we evaluated the possibility that we can utilize hair shaft miR‐29a levels as disease marker of scleroderma. Hair samples were obtained from 20 scleroderma patients, five dermatomyositis patients and 13 controls. microRNAs were purified from hairs as well as skins or sera, and miR‐29a levels were measured with quantitative real‐time polymerase chain reaction. Mean hair miR‐29a levels in scleroderma patients were significantly lower than those in control subjects or dermatomyositis, while expression levels of hair shaft marker keratin 34 were similar among them. There was no strong correlation among the miR‐29a levels in the hair, skin and serum of each patient, suggesting that hair microRNAs can be independent biomarkers. We found scleroderma patients with decreased miR‐29a levels had contracture of the phalanges at a significantly higher prevalence than those without. To confirm the clinical usefulness of hair microRNAs, large‐scale researches are needed in the future.

Evaluation of sentinel node biopsy for cutaneous squamous cell carcinoma.

Sentinel lymph node biopsy (SLNB) is a standard care for cutaneous melanoma but its role in cutaneous squamous cell carcinoma (SCC) has not been established. Clinical data was obtained from 54 patients with SCC who received SLNB with the usage of blue dye and radioisotope colloid methods. The positive rate of SLNB in SCC was 7.4%. If the cases were limited to more than T2, the positive rate was 12.9%. Three of 41 patients who was estimated negative LN metastasis by the preoperative tests had micrometastasis (7.3%). Among 13 patients who were suggested to have metastasis in the preoperative tests, only one patient had histological metastasis. One patient with SCC located in the lower lip showed negative SLNB and subsequently developed node recurrence. In conclusion, the efficacy of SLNB in SCC is comparable to that of melanoma in the positive rate. There are two kinds of benefit, avoidance of unnecessary complete lymph node dissection and early detection of metastasis.

Expression of Matrix Metalloproteinase-13 Is Controlled by IL-13 via PI3K/Akt3 and PKC-δ in Normal Human Dermal Fibroblasts

IL-13, a T helper type 2 cytokine, is reported to be increased in the tissue of patients with atopic dermatitis (AD). In addition, chronic lichenified plaques in AD show thickened epidermis and dermis. We hypothesized that IL-13 is involved in tissue remodeling by altering the expression of matrix metalloproteinases (MMPs). In this study, we examined the MMP-related genes targeted by IL-13 in human dermal fibroblasts using a complementary DNA microarray. We focused on the MMP-13 gene, which was identified as one of the MMPs suppressed by IL-13. IL-13 downregulated both MMP-13 protein and mRNA expression. IL-13 suppressed MMP-13 expression more effectively in the presence of protein kinase C (PKC)-δ inhibitor, whereas IL-13 upregulated MMP-13 in the presence of inhibitors of phosphoinositide 3-kinase (PI3K)/Akt pathway or Akt3-specific small interfering RNA. Our results suggest that MMP-13 expression is negatively controlled by PI3K/Akt3 and positively regulated by PKC-δ in the presence of IL-13. Taken together, these findings indicate that IL-13 may induce the formation of thickened dermis in AD by decreasing collagen degradation. Blockade of IL-13 signaling cascades in AD patients may be a new therapeutic approach.

Submental perforator flap: location and number of submental perforating vessels.

The sites and numbers of submental perforator vessels were examined using a Doppler probe in 21 volunteers. The subcutaneous vascular network from each perforator was studied in three cases of dissection of upper-neck lymph nodes among the volunteers. A perforator from the submental vessels was noted in all 21 volunteers: a single perforator in 13 cases, and double perforators in eight. The main perforator, which had some subdermal branches, was located 31.8 (8.3) mm in front of the facial artery that was palpated at the mandible. Five patients who presented with skin defects on the cheek and the chin had the submental perforator flap reconstructed, excluding the platysma muscle. All flaps covered the wounds. The submental perforator flap was useful for reconstructing skin defects on the cheek and the chin, because the site of the submental perforator was stable and raising the flap was easy, and the colour and texture matches were acceptable.

miR-205 down-regulation promotes proliferation of dermatofibrosarcoma protuberans tumor cells by regulating LRP-1 and ERK phosphorylation

Dermatofibrosarcoma protuberans (DFSP) is an intermediate malignancy of the skin. Although COL1A1/PDGFB fusion gene was identified in the tumor cells recently, not all of the cases were positive for the fusion gene, and further researches are still needed to clarify the pathogenesis of DFSP. In this study, we investigated the role of microRNAs in the tumor. microRNA PCR array showed several microRNAs increased or decreased in DFSP in vivo compared with dermatofibroma (DF) and normal skin. Among them, the expression of miR-205 was down-regulated in DFSP compared with DF and normal skin. In situ hybridization showed that miR-205 expression was evident in dermal fibroblasts of normal skin although hardly detected in tumor cells of DF or DFSP. miR-205 inhibitor increased cell proliferation and the luciferase activity of 3′UTR of low-density lipoprotein receptor-related protein-1 (LRP-1) in cultured normal dermal fibroblasts. Immunohistochemistry showed the expression of LRP-1 was increased in DFSP tissue. Knockdown of LRP-1 suppressed cell growth and down-regulated extracellular signal-regulated kinase (ERK) phosphorylation without affecting MEK phosphorylation in cultured DFSP cells. Taken together, LRP-1 overexpression caused by the miR-205 down-regulation may play a role in the abnormal proliferation of DFSP cells via directly regulating ERK phosphorylation.

The expression of HER-2 in extramammary Paget's disease

Extramammary Pagets disease (EMPD) is a rare intraepidermal adenocarcinoma. The common sites of EMPD involvement are the vulva, perineal, perianal, scrotal and penile skin. Several studies have shown that HER-2/neu, also known as c-erbB-2, is amplified and overexpressed in many cancers. In this study, we investigated the expression and clinical significance of HER-2 in Japanese patients with EMPD. Keratinocytes in epidermis were slightly positive for HER-2. As for EMPD, 19 of 31 EMPD were positive for HER-2 (61%). There is significant correlation between the presence of invasion and strong positivity (3+) for HER-2 (p < 0.02). Furthermore, there is significant correlation between the presence of lymph node metastasis and strong positivity (3+) for HER-2 (p < 0.02). These results suggest that patients with EMPD strongly positive for HER-2 may have high risk for lymph node metastasis and should be followed up carefully. The observed overexpression of HER-2 in EMPD presents a potential therapeutic target for adjuvant treatment of this disease. Treatment with trastuzumab is well established in breast cancer with HER-2 overexpression and is recommended by several consensus statements. The results of the present study indicate that targeting therapies for HER-2, such as trastuzumab, may be used for EMPD particularly in patients with invasive and/or metastatic EMPD.

Down-regulation of microRNA-196a in the sera and involved skin of localized scleroderma patients

BackgroundLocalized scleroderma (LSc) exhibits fibrosis of the skin and subcutaneous tissue. LSc shows an excessive deposition of type 1 collagen.ObjectivesTo elucidate the mechanism of type 1 collagen overexpression in LSc, we investigated the epigenetics, focusing on microRNA (miRNA).Materials & MethodsmiRNA expression profile was determined by PCR array analysis. The expression of microRNA-196a (miR-196a) in the skin tissuewas examined by in situ hybridization or real-time PCR. The serum levels of miR-196a were measured by real-time PCR.ResultsPCR array analysis demonstrated that the miR-196a level was markedly decreased in LSc skin tissue in vivo. The transfection of specific inhibitor for miR-196a into normal cultured human dermal fibroblasts led to the up-regulation of type 1 collagen protein in vitro. Furthermore, the serum levels of miR-196a were significantly decreased in LSc patients.ConclusionDown-regulation of miR-196a and subsequent overexpression of type 1 collagen in dermal fibroblasts may play a key role in the pathogenesis of LSc. The serum levels of miR-196a may be useful as a diagnostic marker of LSc.

Up-regulated type I collagen expression by the inhibition of Rac1 signaling pathway in human dermal fibroblasts

Tissue remodeling is known to play important roles in wound healing. Although Rac1 is reported to be one of the key signaling molecules in cutaneous wound healing process, the exact mechanisms of Rac1-mediated tissue remodeling is still unknown. This study investigated the role of Rac1 in the regulation of extracellular matrix in cultured human dermal fibroblasts obtained by skin biopsy from three healthy donors. Protein levels of type I collagen in cultured human fibroblasts were increased by the treatment with Rac1 inhibitor NSC23766 in a dose-dependent manner. However, the mRNA levels of alpha2(I) collagen was not altered by the inhibitor. On the other hand, by the addition of inhibitor, half-lives of type I collagen protein were increased and MMP1 levels were reduced. These data suggest that blockade of Rac1 signaling results in accumulation of type I collagen due to decreased collagenase activity. This study also suggests that controlling Rac1 signaling is a new therapeutic approach to chronic/untreatable ulcer.