Young-Wook Ryoo

Melatonin reduces ultraviolet-B induced cell damages and polyamine levels in human skin fibroblasts in culture

UV radiation is known to cause photoaging of the skin and is considered one of the leading cause of developing skin carcinogenesis. Melatonin which has a highly lipophilic molecular structure facilitating penetration of cell membranes and serving as an extra- and intracellular free radical scavenger has been demonstrated to protect photodamage of skin affected by UV exposure. In this study, we have examined the role of melatonin in response to UVB induced photodamaging process, using human skin fibroblasts in vitro. Cell survival curves after UVB irradiation showed dose-dependent decrease. Only 60% of fibroblasts were survived at 140 mJ/cm2 UVB irradiation. By pre-cultivation of cells with melatonin (100 nM), a significant number of cells remained unaffected. After UVB irradiation with 70 mJ/cm2, the level of putrescine was 1.7±0.3 fold increased compared to melatonin pre-treated group. In Northern analyses, the transcriptional level of ornithine decarboxylase (ODC) gene expression was increased by UVB irradiation and prohibited by melatonin. These results indicated that melatonin was effectively able to neutralize membrane peroxidation when present in relevant concentration during UVB irradiation and diminishes the UVB-induced increase of polyamine synthesis and ODC gene expression. Collectively, ODC response to UVB induced changes are possibly involves a melatonin or antioxidant sensitive regulatory pathway in normal human skin fibroblast.

Clinical Improvement of Striae Distensae in Korean Patients Using a Combination of Fractionated Microneedle Radiofrequency and Fractional Carbon Dioxide Laser

BACKGROUND Striae distensae are dermal scars with flattening and atrophy of the epidermis. OBJECTIVE To evaluate the efficacy and safety of combination therapy with fractionated microneedle radiofrequency (RF) and fractional carbon dioxide (CO2) laser in the treatment of striae distensae. MATERIALS AND METHODS Thirty patients (30 female; mean age 33, range 21–51, Fitzpatrick skin type IV) with moderate to severe striae distensae were enrolled in this study. Patients were divided into three groups: fractional CO2 laser only (n = 10), microneedle RF only (n = 10), and combination (n = 10). RESULTS Improvement was evaluated using a visual analogue scale (range 1–4). Mean clinical improvement score of the dermatologist was 2.2 in the fractional CO2 laser–treated group, 1.8 in the microneedle RF–treated group, and 3.4 in the combination group. Through skin biopsy, we observed thickened epidermis and a clear increase in the number of collagen fibers in the microneedle RF– and fractional CO2 combination–treated sites. Consistent with these results, greater expression of transforming growth factor‐&bgr;1 and stratifin was observed in treated sites. CONCLUSION Combination therapy of fractionated microneedle RF and fractional CO2 laser is a safe treatment protocol with a positive therapeutic effect on striae distensae.

Characterization of mutations of the type VII collagen gene (COL7A1) in recessive dystrophic epidermolysis bullosa mitis (M-RDEB) from three Korean patients

In recent years, the molecular basis for the main subtypes of epidermolysis bullosa (EB) has been elucidated with pathogenetic mutations delineated in ten different genes encoding structural components of the dermal-epidermal junction. Both the autosomal dominant and recessive forms of dystrophic EB (DEB) is caused by mutations in the COL7A1 gene. Type VII collagen is a major component of anchoring fibrils, structural elements that stabilize the attachment of the basement membrane to underlying dermis. Recent delineation of the exon-intron organization of the COL7A1 gene provided the basis for the comprehensive design of PCR primer pairs that amplified exons in genomic DNA by placing the primers on the flanking introns. A number of COL7A1 mutations have been reported and some genotype-phenotype correlations are starting to emerge. In this study, we examined mutational analyses from three Korean patients with recessive dystrophic EB (RDEB) mitis. We designed and optimized primers according to the previously reported sequences. Such PCR amplification products can be examined by electrophoretic scanning technique, CSGE heteroduplex analyses. Utilizing heteroduplex analyses, we have identified a number of sequence variants in COL7A1 both in unaffected individuals and in patients with M-RDEB. Mutation detection of the COL7A1 gene revealed six allelic mutations (V6677E, P6685S, Y3749S, P6084S, P6695R and G6697C). We suggest that the full length of type VII collagen polypeptide are synthesized, but those missense mutations, that may affect a critical amino acid, can alter the conformation of the protein and interferes with the assembly and packing of type VII collagen molecules into anchoring fibrils. Immunohistochemical study of skin biopsies by use of anti-type VII collagen antibody showed markedly reduced staining and presence of a dermo/epidermal cleavage. This is the first report of a COL7A1 mutation study in DEB from Korean patients. We hope that these data contribute to the expanding database on COL7A1 mutations in dystrophic epidermolysis bullosa, and further illustrate the extensive diversity of mutational events that led to the RDEB phenotype.

ALL-TRANS-RETINOIC ACID DOWN-REGULATES ELASTIN PROMOTER ACTIVITY ELEVATED BY ULTRAVIOLET B IRRADIATION IN CULTURED SKIN FIBROBLASTS

Topical tretinoin therapy produces clinical improvements in the fine wrinkling of photodamaged skin, possibly by enhancement of collagen synthesis. A major biochemically and histologically detectable change in photodamaged skin is the accumulation of abnormal elastic fibers (elastotic material). However, little is known about the effects of retinoic acid and ultraviolet B (UVB) on elastin gene expression. Consequently, we examined the effects of all-trans-retinoic acid (t-RA) and UVB on elastin gene expression in cultured human skin fibroblasts in vitro. Elastin mRNA gene expression was up-regulated in response to UVB by approximately equal to 3-fold, in a dose dependent manner, between 3 and 10 mJ/cm2 doses. Similar results were obtained by chloramphenicol acetyltransferase assay, in which a maximal promoter activation more than 5.4-fold that in nonirradiated controls occurred after a single dose of 20 mJ/cm2. Also t-RA inhibited the increase in elastin mRNA level following a single exposure to UVB by approximately 16%, and the increase in promotor activity by about 65%. The inhibitory effect of t-RA on elastin induced by UVB was also demonstrated by indirect immunofluorescence studies. Taken together, t-RA down-regulated human elastin gene expression elevated by a single exposure to UVB at transcriptional and possibly protein levels. These results suggest that the anti-photoaging effect of t-RA may be related, at least in part, to down-regulation of elastin gene expression elevated by UVB.

Interferon-γ upregulates the stromelysin-1 gene expression by human skin fibroblasts in culture

The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-γ (IFN-γ) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-γ treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-γ stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-β (TGF-β) showed the antagonistic action to the effects of IFN-γ in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-γ. These data suggest that IFN-γ is an up-regulator and TGF-β is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.