Identifi cation of the Underlying Genetic Factors of Skin Aging in a Korean Population Study.

Genetic polymorphisms may affect the molecular mechanisms underlying determination of skin type. So far, several genetic studies have been reported; however, very few studies have been conducted to examine the relationship between genotype and skin phenotypes. In this study, the genome sequences of individuals tested for five cosmetic characteristics (wrinkles, moisture content, pigmentation, oil content, and ensitivity) were determined, and we also conducted five genome-wide association studies (GWASs) to identify predictive markers. Some single-nucleotide polymorphisms (SNPs) within those genes were more frequent in individuals exhibiting stronger traits. GWASs revealed that two genome-wide significant SNPs within FCRL5 and OCA2 genes were associated with wrinkles and pigmentation, respectively (p < 5 × 10-8), and that genomewide SNPs in 21 genes (wrinkles: FCRL5, REEP3, ADSS, and SPTLC1; moisture: TBX4, TRPM3, CEMIP2, CTSH, and TTC39C; pigmentation: OCA2, NCLN, TNS1, CDC42BPA, HS3ST4, and UNCX; oil: SYN2, CNDP1, GAS6, INSR, and TNFRSF19; and sensitivity: CREB5) might be associated with five skin phenotypes. Among these, a genome-wide significant SNP (rs117381658) and the SNP located downstream of FCRL5, which encodes a member of the immunoglobulin receptor family, were associated with an increased risk of wrinkles (p = 1.52 × 10-8). The other genome-wide significant SNP (rs74653330) was associated with a decreased risk of pigmentation (p = 1.04 × 10-8), which is located in the coding region of OCA2 that encodes for a transporter of melanin. Our study reports genetic factors associated with skin cosmetology parameters in the Korean population. We hope our findings will provide a foundation for further genetic and molecular studies related to custom cosmetics. Based on these findings, the industry will be able to provide consumers with ingredients capable of palliating the lack of function associated in genes with SNPs.

Anti-Wrinkle Benefits of Peptides Complex Stimulating Skin Basement Membrane Proteins Expression.

The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.

Quantitative monitoring of laser-treated engineered skin using optical coherence tomography.

Nowadays, laser therapy is a common method for treating various dermatological troubles such as acne and wrinkles because of its efficient and immediate skin enhancement. Although laser treatment has become a routine procedure in medical and cosmetic fields, the prevention of side-effects, such as hyperpigmentation, redness and burning, still remains a critical issue that needs to be addressed. In order to reduce the side-effects while attaining efficient therapeutic outcomes, it is essential to understand the light-skin interaction through evaluation of physiological changes before and after laser therapy. In this study, we introduce a quantitative tissue monitoring method based on optical coherence tomography (OCT) for the evaluation of tissue regeneration after laser irradiation. To create a skin injury model, we applied a fractional CO2 laser on a customized engineered skin model, which is analogous to human skin in terms of its basic biological function and morphology. The irradiated region in the skin was then imaged by a high-speed OCT system, and its morphologic changes were analyzed by automatic segmentation software. Volumetric OCT images in the laser treated area clearly visualized the wound healing progress at different time points and provided comprehensive information which cannot be acquired through conventional monitoring methods. The results showed that the laser wound in engineered skins was mostly recovered from within 1~2 days with a fast recovery time in the vertical direction. However, the entire recovery period varied widely depending on laser doses and skin type. Our results also indicated that OCT-guided laser therapy would be a very promising protocol for optimizing laser treatment for skin therapy.

Different effects of five depigmentary compounds, rhododendrol, raspberry ketone, monobenzone, rucinol and AP736 on melanogenesis and viability of human epidermal melanocytes.

Numerous medications are used to treat hyperpigmentation. However, several reports have indicated that repeated application of some agents, such as rhododendrol (RD), raspberry ketone (RK) and monobenzone (MB), can be toxic to melanocytes. Although these agents had severe side effects in human trials, no current in vitro methods can predict the safety of such drugs. This study assessed the in vitro effects of five depigmentary compounds including leukoderma-inducing agents. In particular, we determined the effects of different concentrations and exposure times of different depigmentary agents on cell viability and melanogenesis in the presence and absence of ultraviolet B (UVB) radiation. Concentrations of RD, RK and MB that inhibit melanogenesis are similar to concentrations that are cytotoxic; however, concentrations of rucinol (RC) and AP736 that inhibit melanogenesis are much lower than concentrations that are cytotoxic. Furthermore, the concentrations that cause toxic effects depend on exposure duration, and prolonged exposure to RD, RK and MB had more cytotoxic effects than prolonged exposure to RC and AP736. The cytotoxic effects of RD and RK appear to be mediated by apoptosis due to increased expression of caspase-3 and caspase-8; UVB radiation increased the cytotoxicity of these agents and also increased caspase activity. Our results indicate that different leukoderma-inducing compounds have different effects on the viability of normal epidermal melanocytes and suggest that the in vitro assay used here can be used to predict whether an investigational compound that induces leukoderma may lead to adverse effects in human trials.

Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS-ERK activation.

Mitochondrial dynamics control mitochondrial functions as well as their morphology. However, the role of mitochondrial dynamics in melanogenesis is largely unknown. Here, we show that mitochondrial dynamics regulate melanogenesis by modulating the ROS-ERK signaling pathway. Genetic and chemical inhibition of Drp1, a mitochondrial fission protein, increased melanin production and mitochondrial elongation in melanocytes and melanoma cells. In contrast, down-regulation of OPA1, a mitochondria fusion regulator, suppressed melanogensis but induced massive mitochondrial fragmentation in hyperpigmented cells. Consistently, treatment with CCCP, a mitochondrial fission chemical inducer, also efficiently repressed melanogenesis. Furthermore, we found that ROS production and ERK phosphorylation were increased in cells with fragmented mitochondria. And inhibition of ROS or ERK suppressed the antimelanogenic effect of mitochondrial fission in α-MSH-treated cells. In addition, the activation of ROS-ERK pathway by mitochondrial fission induced phosphorylation of serine73 on MITF accelerating its proteasomal degradation. In conclusion, mitochondrial dynamics may regulate melanogenesis by modulating ROS-ERK signaling pathway.

Stiffness-modulated water retention and neovascularization of dermal fibroblast-encapsulating collagen gel.

There is increasing evidence that matrix stiffness modulates various phenotypic activities of cells surrounded by a three-dimensional (3D) matrix. These findings suggest that matrix stiffness can also regulate dermal fibroblasts activities to remodel, repair, and recreate skin dermis, but this has not yet been systematically demonstrated to date. This study examines the effects of matrix rigidity on the morphology, growth rates, and glycosaminoglycan (GAG) production of dermal fibroblasts cultured in collagen-based hydrogels with controlled elastic moduli. The elastic moduli (E) of collagen hydrogels were increased from 0.7 to 1.6 and 2.2 kPa by chemically cross-linking collagen fibrils with poly(ethylene glycol) disuccinimidylester. Increasing E of the hydrogel led to decreases in cellular spreading, nuclear aspect ratio, and growth rate. In contrast, the cellular GAG production level was elevated by increasing E from 0.7 to 1.6 kPa. The larger accumulation of GAG in the stiffer hydrogel led to increased water retention during exposure to air, as confirmed with magnetic resonance imaging. Additionally, in a chicken chorioallantoic membrane, a cell-encapsulating hydrogel with E of 1.6 kPa created dermis-like tissue with larger amount of GAG and density of blood vessels, while a cell-hydrogel construct with E of 0.7 kPa generated scar-like tissue. Overall, the results of this study will be highly useful for designing advanced tissue engineering scaffolds that can enhance the quality of a wide array of regenerated tissues including skin.

Depigmentation effect of kadsuralignan F on melan-a murine melanocytes and human skin equivalents.

The development of melanogenic inhibitors is important for the prevention of hyperpigmentation, and, recently, consideration has been given to natural materials or traditionally used ingredients such as Chinese medicine. The aim of this study is the evaluation of a new anti-melanogenic candidate, kadsuralignan F, from the natural plant Kadsura coccinea, as well as the determination of mechanisms of melanogenesis inhibition at a molecular level. Kadsuralignan F significantly reduced melanin synthesis in a dose-dependent manner in a murine melanocyte cell line and human skin equivalents. There was no direct inhibition on mushroom tyrosinase or cell-extract tyrosinase activity, and mRNA expression of tyrosinase and other melanogenic genes such as tyrosinase-related protein-1 (trp-1) or trp-2 were not affected by kadsuralignan F. Interestingly, the protein level of tyrosinase was dramatically downregulated with kadsuralignan F treatment. We found that a decrease of tyrosinase protein by kadsuralignan F was fully recovered by MG132, a proteasome inhibitor, but not by chloroquine, a lysosome inhibitor. In this study, we found that kadsuralignan F, a lignan from an extract of Kadsura coccinea, has an inhibitory activity on melanin synthesis through tyrosinase degradation. These findings suggest that kadsuralignan F can be used as an active ingredient for hyperpigmentation treatment.

Identification of sake extract as a new anti-melanogenic ingredient by in vitro and clinical trials.

Overproduction of melanin is the cause of skin hyperpigmentation, which is related to several skin diseases and cosmetic concerns. Sake is a Japanese alcoholic beverage produced from rice and water by fermentation, but is little known for its effect on melanogenesis. To identify the effect of sake extract on melanin synthesis, a melanin assay was performed in melan-A murine melanocytes. Sake extract treatment significantly inhibited melanin production in a dose-dependent manner, and tyrosinase, the rate-limiting enzyme of melanogenesis, decreased significantly at the protein level. Further investigations were performed with multiple assay systems; a sake extract reduced melanin production in melan-A/SP-1 murine cell co-culture, and also in MelanoDerm, a skin equivalent model of human keratinocytes-melanocytes. Finally, subjects were treated with a formula containing the sake extract. Topical application of the sake extract product improved skin lightness (L*) significantly within 7 days. We identified sake extract as a new anti-melanogenic ingredient through in vitro and in vivo experiments. These results suggest that a sake extract can be used to improve skin hyperpigmentation.

Activation of transient receptor potential melastatin 8 reduces ultraviolet B-induced prostaglandin E2 production in keratinocytes.

Transient receptor potential melastatin 8 (TRPM8) is a member of the TRP family, and is activated at temperatures below 22°C, or by cooling compounds such as menthol. In this study, it was found that a new role of TRPM8 activation on prostaglandin E2 (PGE2), an inflammatory cytokine and dendritogenesis stimulator of normal human melanocytes. Normal human keratinocytes were pretreated with menthol or incubated at 22°C for TRPM8 activation before ultraviolet (UV)-B irradiation. To examine the specificity between TRPM8 activation and PGE2 release, we inhibited TRPM8 with the antagonist (capsazepine), or introduced TRPM8 siRNA for a gene silencing experiment. UV-B irradiation significantly induced PGE2 release in normal human keratinocytes. Interestingly, activation of TRPM8 at 22°C or with menthol inhibited UV-B-induced PGE2 release. The effect of the TRPM8 agonist was completely blocked by pretreatment with the TRPM8 antagonist, capsazepine. When TRPM8 expression was suppressed by siRNA, UV-B irradiation still upregulated PGE2 in keratinocytes, but pretreatment of menthol or low temperature did not inhibit UV-B-induced PGE2. In conclusion, the activation of TRPM8 inhibits UV-B-induced PGE2 production in keratinocytes, and the activation of TRPM8 may reduce inflammatory responses in skin.

Fabrication of microgel-in-liposome particles with improved water retention.

Corneocytes represents the main water reservoir of stratum corneum, and that ability intimately arises from their architecture and total composition. Here we describe a novel method for fabricating a microgel-in-liposome (M-i-L) structure consisting of a sodium hyaluronate microgel and a lipid membrane envelop in order to mimic corneocyte cell structures. The essence of our approach is to use a lecithin-based microemulsion with a very low interfacial tension between the water droplet and oil continuous phase. Using this emulsion enables us to stabilize a dispersion of microgel particles without phase separation or aggregation. The addition of excess water produced single-core or multicore microgel particles enveloped in a lipid layer. To demonstrate the applicability of this unique vesicle system, we encapsulated a high concentration of natural moisturizing factor (NMF) in the microgel core and investigated how the M-i-L structure affected the water retention in comparison with other control systems. We have observed that our M-i-L particles with the NMF in the core, which mimicked the corneocyte cell structure, showed an excellent ability to retain water in the system. This experimental result inspired us to investigate how corneocyte cells, which feature a lipid-enveloped hydrogel structure, provide such long-lasting hydration to the skin.

Alternative evaluation method in vitro for the water-resistant effect of sunscreen products.

BACKGROUND/PURPOSE: Sunscreen products today represent a trend of providing not only simple sun protection factor (SPF)/protection of UVA (PFA) but also other additional benefits. For example, as popularized by seasonless use of sunscreens, the special function of water resistance or sand proof is added to sunscreens as well as for leisure. Because a human in vivo test is time consuming and expensive, a screening process has been tried using an accurate in vitro system. In this study, we suggest the development of an in vitro test that can predict the result of in vivo water resistance of sunscreens. METHODS: Water resistance is presented as a comparison of initial SPF and water-exposed SPF by immersion and washing. In order to be comparable with the in vivo test, water immersion and flow were defined as the basic statements. Also, substrate, revolutions per minute (r.p.m.)--rotative velocity--of propeller inducing water flow, and time of immersion were defined as controlled factors. Considering the strength, separation of test material and adhesive texture, a PMMA plate was selected as suitable among commercial substrates: Transpore tape, VITRO SKIN, and PMMA plate. Also, when the PMMA plate was adhered on the wall of a water bath, the water turbulence of the rotational propeller alone was not strong enough to wash off the test material from the substrate. Therefore, PMMA plates were fixed on the axis. In this experiment, the most important thing is whether this in vitro system can predict correctly. Hence, we tried to match the in vitro water resistance following from our control factors and water resistance value of the in vivo test. RESULTS: We found the immersion time and r.p.m. of controlled factors to obtain the target water resistance using design of experiment, MiniTab statistical package. Response optimization yielded the optimal in vitro conditions of 150 r.p.m./60 min. The repeatability and reproducibility of this in vitro system were also good in validation studies. CONCLUSIONS: This study enables to modify an in vivo water resistance test and predict the result of in vivo water resistance by the manufacture of effective equipment and choosing a suitable substrate. Compared with in vivo results, our in vitro system is more time and cost effective, and provides reliable results.

Influence of polyol and oil concentration in cosmetic products on skin moisturization and skin surface roughness.

BACKGROUND/PURPOSE: The purpose of this study is to investigate the optimum combination of polyols and oils in moisturizing cosmetic products to improve the human skin moisturization and skin surface roughness. Polyols and oils are essential ingredients in skin care products, but it is still not understood how their concentrations affect their efficacy and sensory properties on human skin. We investigated the effect of polyol and oil concentration on skin properties by noninvasive methods. METHODS: The polyols consisted of glycerin and butylenes glycol in a ratio of 1:1 and the oils consisted of equal parts of hydrogenated polydecene, cethyl ethylhexanoate and pentaerythrityl tetraethylhexanoate. All cosmetic products were made in O/W emulsions in concentrations ranging from 0% to 30% for polyols and from 0% to 35% for oils. We investigated the effect on water content and skin surface roughness on the forearm after application of the cosmetic products. The skin water contents were measured by a Corneometer CM825 and the skin surface roughness by visual coring of skin surface biopsies in the scanning electron micrographs. RESULTS: In the first study, we found that the water content of the skin correlated highly with the polyol (up to 30%) and oil (up to 12%) concentrations, respectively. At two hours after application, the correlation coefficients were 0.971 and 0.985, respectively (P<0.01). Skin surface roughness not only showed a strong concentration dependence on polyols and oils (up to 6%). In the second study, we investigated the optimum combination of polyols and oils to improve the skin moisturization and skin surface roughness by the Response surface methodology. The water content of the skin surface was high in the ratio of polyol to oil (30:12 and 25:30). The skin surface roughness was improved considerably in the ratio of polyols to oil (30:6 and 30:35). CONCLUSIONS: Our results indicated that the skin surface properties were improved in the different ratios of their concentrations because they are influenced by not one ingredient but the interaction between polyols and oils. In this study, we could recommend the optimum concentration of polyols and oils to improve the skin surface properties. Further studies will be performed with other ingredients such as surfactants, lipids and so on.

Comparison and correlation between stinging responses to lactic acid and bioengineering parameters.

In evaluating the safety of a novel cosmetic product or a new chemical, it is important to assess susceptible population. One group of subjects is known to stingers who are more likely to experience sensory effects such as stinging and burning after contacting with cosmetics. The purpose of the study is to measure skin biophysical parameters noninvasively in stingers and non-stingers and to see their correlations with stinging responses. 298 women were evaluated by modified lactic acid stinging test with 5% lactic acid solution rather than classic 10% solution because of strong reaction in Asian populations. Transepidermal water loss (TEWL), skin hydration, sebum content, and pH were measured using the bioengineering instruments in an environment-controlled room. Correlations between stinging responses and skin biophysical parameters were statistically analysed. There was a positive correlation between stinging responses and TEWL evaluation. However, no correlations was observed between stinging responses and other parameters such as skin hydration, sebum content, and pH. Our data indicate that there is a relationship between the degree of stinging and the skin barrier function. However, we believe that various additional studies are necessary to characterize skin of stingers and the pathogenesis.

Correlation between a Cutometer and quantitative evaluation using Moire topography in age-related skin elasticity.

BACKGROUND/PURPOSE: As aging occurs, our skin gets more wrinkles, becomes drier and loses its elasticity. Validating the evaluation of skin elasticity is especially important, because it is not as visible as other signs of aging such as wrinkles. So it is needed that the method for measuring skin elasticity is able to reflect perception about the change of the skin state. METHODS: Here, the correlation between age and the parameters given by a Cutometer is identified and the main parameters that reflect the decreases in skin elasticity in terms of ages are presented. Also, Moire's system, an evaluation method to quantify the sensory value of viewing, is developed. A five-grade standard of Moire topographic photo scale on the face is used to evaluate the state of skin elasticity and lifting 20- to 61-year-old women. Based on this photo standard, scoring is performed using a five-grade system by three specialists to obtain the consensus score. The score is compared with the result of a Cutometer. RESULTS: Significant negative correlations between age and results of a Cutometer (r=-0.687-0.725), Moire's topography scores (r=-938), were found. Some Cutometer parameters and the decreases in skin elasticity in terms of ages were highly correlated (r=-0.687-0.725). The results from Moire system and flexibility as sensory evaluation also had a very high correlation with age (r=-0.765-0.932). Finally, we have shown the significance of the correlation between the result of a Cutometer and the score of Moire topography (r=0.711). CONCLUSIONS: It is considered that Cutometer parameters R7 and R2 are used as main parameters to assess skin elasticity and aging. And our studies using Moire topography on the face have confirmed that instrumental measurements reflect the decrease in skin elasticity, which is perceived visually.