The effects of the melatonin on ultraviolet-B irradiated cultured dermal fibroblasts

It has been reported that reactive oxygen species (ROS) and oxygen-derived free radicals are generated by ultraviolet (UV) radiation and various chemicals and their important roles in cellular damage and apoptosis are being increasingly recognized. Melatonin is a hormone with multiple functions in humans, produced by the pineal gland and stimulated by beta-adrenergic receptors. Melatonin has been shown to have photo protection properties, but there has been little progress toward identifying the specific mechanisms of its action. To clarify the role of melatonin as a free radical scavenger, in response to ultraviolet-B (UVB) irradiation, we investigated the effects of UVB and melatonin on cytotoxicity, lipid peroxidation, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling (TUNEL) assay and alteration of cell cycle in cultured skin fibroblast. Cell survival curves after UVB irradiation showed dose dependent decrement pattern by trypan blue exclusion assay. Only 56% of dermal fibroblasts were survived at 140 mJ/cm2 UVB irradiation. The damage was associated with cell membrane lipid peroxidation, as shown by accumulation malondialdehyde (MDA). By pre-cultivation with melatonin (10(-9) M), a significant preventive effect was noted on the increase in the absolute number of surviving cells (up to 92.5% of cells were survived) and the levels of MDA were markedly decreased. These finding suggest significant correlation between an increase of lipid peroxide and cell viability. Morphological changes associated with apoptotic cell death were easily distinguished by TUNEL stain. Quantitative analysis of DNA content of skin fibroblasts was evaluated by flow cytometric analysis performed after vital staining with propidium iodide. UVB suppresses the G1 progression induced pre-G1 arrest leading to apoptotic changes of dermal fibroblast and those are blocked by melatonin pre-treatment. The results show the photodynamic effects of UVB that supposes the production of ROS and arrest the cell cycle. Melatonin, which have newly accepted as a potential UV protection properties, is effective membrane peroxidation inhibitor and prevent the pre-G1 arrest when present in relevant concentration during UVB irradiation.     

UVA irradiation induces collagenase in human dermal fibroblasts in vitro and in vivo

Summary We report the effect of UVA irradiation on collagen metabolism of fibroblasts, including both synthesis of the collagen degrading enzyme collagenase and de novo synthesis of type I collagen as the major structural component of the dermis. For this purpose confluent fibroblast monolayers were irradiated under standardized conditions (5, 15, 35, 60 J/cm² using UVASUN 3000, Mutzhas, Munich, FRG, and UV source Sellas sunlight type 2.001, Sellas, Gevelsberg, FRG). Subsequently, total RNA was isolated and subjected to dot blot and northern blot analysis using oligolabelled cDNA clones for human type I collagen, collagenase and -actin. Collagen type I and -actin mRNA levels remained unaltered following irradiation, suggesting that the synthetic pathway of collagen metabolism at the pretranslational level is not affected by short-term UVA irradiation. However, collagenase mRNA was found to be dose-dependently induced in fibroblasts after irradiation, thus probably contributing to the actinic damage to the dermis. These in vitro data were confirmed in vivo using in situ hybridization on frozen sections of biopsy material obtained from UVA irradiated patients.     

The binding of 8-methoxypsoralen to nuclear DNA of UVA irradiated human fibroblasts in vitro.

Human fibroblasts were exposed to tritium-labelled 8-methoxypsoralen (8-MOP) and longwave ultraviolet (UVA) light in vitro. The proportions of photochemically bound 8-MOP in the nucleus and cytoplasm were studied by autoradiography. The results show that UVA-irradiation of the cells in the presence of 8-MOP gives rise to a significant dose-dependent binding of 8-MOP in the cell nucleus.     

UVA1对人工皮肤成纤维细胞光损伤的影响

目的探讨不同UVA1照射剂量对人工皮肤真皮细胞形态及细胞活性的影响,为建立人工皮肤光损伤模型提供依据。方法以不同剂量UVA1（0J／cm^2-80J／cm^2）照射人工皮肤,通过HE染色,从形态学上观察经UVA1照射后真皮成纤维细胞数量的变化,通过MTT法检测真皮成纤维细胞的活性。结果HE染色示真皮成纤维细胞的数量随UVA1剂量的增大而减少,于真皮浅层较为明显;MTT法示与0J／cm^2组相比,20J／cm^2和30J／cm^2UVA1照射人工皮肤时成纤维细胞活性未受到明显的抑制（P〉0．05）．UVA1剂量大于40J／cm^2时细胞活性下降明显（P〈0．05,P〈0．001）,呈剂量依赖性。结论UVA1照射剂量大于40J／cm^2是造成人工皮肤真皮成纤维细胞光损伤的始剂量。     

Smads regulate collagen gel contraction by human dermal fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction that is implicated in efficient wound healing. The Smad family of proteins mediates signal transduction of the TGF-beta superfamily. However, its role in fibroblast contraction remains unclear. OBJECTIVES: To determine whether Smad proteins regulate fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with human dermal fibroblasts infected with adenoviruses carrying Smads. RESULTS: Overexpression of Smad3, a major signal transducer in the Smad family, enhanced collagen gel contraction by fibroblasts when compared with fibroblasts overexpressing a control lacZ. Addition of a very low concentration of TGF-beta1 that did not affect the collagen gel contraction by itself enhanced the contraction by fibroblasts overexpressing Smad3. In contrast, TGF-beta1-mediated collagen gel contraction was suppressed by overexpression of Smad7, a major inhibitory regulator in the Smad family, in fibroblasts. In addition, inhibitors of the Erk and p38 pathways, PD98059 and SB203580, did not affect TGF-beta1-mediated collagen gel contraction by dermal fibroblasts. CONCLUSIONS: Modulation of Smad3 or Smad7 expression in dermal fibroblasts affected their contraction of collagen gels possibly by regulating TGF-beta signalling in fibroblasts.     

绵头雪莲花提取物对UVA照射后人成纤维细胞增殖的影响

目的:明确绵头雪莲花提取物对UVA照射下的人成纤维细胞的保护作用。方法:制备绵头雪莲花浸膏,将培养的人成纤维细胞分为正常对照组、UVA照射组(10 J/cm~2)及UVA+药物组,CCK8法检测细胞增殖活性,并比较各组吸光度值。结果:UVA+药物组漂浮死细胞(低于5%)少于UVA照射组(约20%~30%);UVA+药物组(200μg/mL)吸光度值为1.16±0.22,高于UVA照射组的0.5±0.12,差异有统计学意义(P0.05)。结论:绵头雪莲花提取物对UVA照射人成纤维细胞具有一定的保护作用。     

UVB对皮肤成纤维细胞相关骨架蛋白表达的影响

目的:确定UVB对皮肤成纤维细胞骨架蛋白的影响.方法:用150 mJ/cm2的UVB照射培养的皮肤成纤维细胞,用MTT法检测细胞活性,用Western blot法检测α-微管蛋白、β-微管蛋白、β-肌动蛋白和波形蛋白的含量.结果:150 mJ/cm2 UVB照射后β-微管蛋白含量逐渐降低,波形蛋白逐渐升高,α-微管蛋白和β-肌动蛋白改变不明显.结论:在UVB诱导人皮肤成纤维细胞光损伤过程中,β-微管蛋白和波形蛋白可能发挥着重要作用.     

Influence of human dermal fibroblasts on epidermalization

Using a method that allowed the reconstruction of simplified living human skin in vitro, we investigated the effects of collagen texture and dermal fibroblasts on epidermal growth. Like in vivo skin, our in vitro model comprised two tissues: a dermal equivalent and an overlying epidermis. It permitted measurement of epidermal growth and therefore evaluation of the effect of the dermal equivalent on this growth. Epidermal growth was enhanced when the collagen matrix had previously been reorganized by fibroblasts, and was greatest when living fibroblasts persisted in this matrix. On cell-free collagen gel and on collagen matrices containing dead fibroblasts, epidermal growth increased when the medium was conditioned by fibroblasts grown in monolayers. We conclude that the function of the fibroblasts is not only to synthesize and degrade the extracellular matrix, but also to regulate epidermalization; on the one hand by remodeling the collagen fibers, and on the other by secreting diffusible factors that promote epidermal growth. These results underline the importance of fibroblasts in dermo-epidermal interactions, and show that the skin equivalent culture model provides a way to quantitatively study these interactions.     

Pentosidine in advanced glycation end-products (AGEs) during UVA irradiation generates active oxygen species and impairs human dermal fibroblasts

Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.     

Multilineage differentiation potential of human dermal skin-derived fibroblasts

Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14(-), CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+) in well-established adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-cell-like cells expressed vimentin, fibronectin and collagen; they were less positive for alpha-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.     

Redox regulation of cellular stress response by ferulic acid ethyl ester in human dermal fibroblasts: role of vitagenes

Skin is one of the main targets for reactive oxygen species; thus, reactive oxygen species-induced damage and protein and lipid modifications occur, and skin can undergo a wide array of diseases, from photosensitivity to cancer. In this study, human dermal fibroblasts exposed to hydrogen peroxide (0-1000 micromol/L) exhibited a marked increase in both protein carbonyls and 4-hydroxy-2-nonenal, which are indices of protein and lipid oxidation, respectively. An amount of 25 micromol/L ferulic acid ethyl ester, a well-known nutritional antioxidant, significantly counteracted both protein and lipid oxidation and reduced the loss in cell viability elicited by 500 micromol/L of hydrogen peroxide. A common way for cells to react to oxidative stress is up-regulation of vitagenes. To the vitagene family belong the heat shock proteins heme oxygenase-1 and heat shock protein-70, which are involved in the cellular defense against oxidative stress by different mechanisms. The administration of 25 micromol/L ferulic acid ethyl ester significantly decreased hydrogen peroxide-induced protein and lipid oxidation. Dermal fibroblasts exposed to 25 micromol/L ferulic acid ethyl ester in the presence of 500 micromol/L hydrogen peroxide showed an increased level of both heme oxygenase-1 and heat shock protein-70 compared with dermal fibroblasts treated with hydrogen peroxide alone. These findings provide evidence for the protective role of vitagenes in free radical-induced skin damage and highlight the potential protective use of nutritional antioxidants, such as ferulic acid and its derivatives.     

UVA radiation impairs phenotypic and functional maturation of human dermal dendritic cells

There is now strong evidence that the ultraviolet A (UVA) part of the solar spectrum contributes to the development of skin cancers. Its effect on the skin immune system, however, has not been fully investigated. Here, we analyzed the effects of UVA radiation on dermal dendritic cells (DDC), which, in addition, provided further characterization of these cells. Dermal sheets were obtained from normal human skin and irradiated, or not, with UVA at 2 or 12 J per cm2. After a 2 d incubation, the phenotype of emigrant cells was analyzed by double immunostaining and flow cytometry. Results showed that migratory DDC were best characterized by CD1c expression and that only few cells co-expressed the Langerhans cell marker Langerin. Whereas the DC extracted from the dermis displayed an immature phenotype, emigrant DDC showed increased expression of HLA-DR and acquired co-stimulation and maturation markers. We showed here that UVA significantly decreased the number of viable emigrant DDC, a process related to increased apoptosis. Furthermore, UVA irradiation impaired the phenotypic and functional maturation of migrating DDC into potent antigen-presenting cells, in a concentration-dependent manner. The results provide further evidence that UVA are immunosuppressive and suggest an additional mechanism by which solar radiation impairs immune response.