(S)-(-)-blebbistatin O-benzoate has the potential to improve atopic dermatitis symptoms in NC/Nga mice by upregulating epidermal barrier function and inhibiting type 2 alarmin cytokine induction.

Atopic dermatitis is a multi-pathogenic disease characterized by chronic skin inflammation and barrier dysfunction. Therefore, improving the skin's ability to form an epidermal barrier and suppressing the production of cytokines that induce type 2 inflammatory responses are important for controlling atopic dermatitis symptoms. (-)-Blebbistatin, a non-muscle myosin II inhibitor, has been suggested to improve pulmonary endothelial barrier function and control inflammation by suppressing immune cell migration; however, its efficacy in atopic dermatitis is unknown. In this study, we investigated whether (S)-(-)-blebbistatin O-benzoate, a derivative of (-)-blebbistatin, improves dermatitis symptoms in a mite antigen-induced atopic dermatitis model using NC/Nga mice. The efficacy of the compound was confirmed using dermatitis scores, ear thickness measurements, serum IgE levels, histological analysis of lesions, and filaggrin expression analysis, which is important for barrier function. (S)-(-)-Blebbistatin O-benzoate treatment significantly reduced the dermatitis score and serum IgE levels compared to those in the vehicle group (p < 0.05). Furthermore, the histological analysis revealed enhanced filaggrin production and a decreased number of mast cells (p < 0.05), indicating that (S)-(-)-blebbistatin O-benzoate improved atopic dermatitis symptoms in a pathological model. In vitro analysis using cultured keratinocytes revealed increased expression of filaggrin, loricrin, involucrin, and ceramide production pathway-related genes, suggesting that (S)-(-)-blebbistatin O-benzoate promotes epidermal barrier formation. Furthermore, the effect of (S)-(-)-blebbistatin O-benzoate on type 2 alarmin cytokines, which are secreted from epidermal cells upon scratching or allergen stimulation and are involved in the pathogenesis of atopic dermatitis, was evaluated using antigens derived from mite feces. The results showed that (S)-(-)-blebbistatin O-benzoate inhibited the upregulation of these cytokines. Based on the above, (S)-(-)-blebbistatin O-benzoate has the potential to be developed as an atopic dermatitis treatment option that controls dermatitis symptoms by suppressing inflammation and improving barrier function by acting on multiple aspects of the pathogenesis of atopic dermatitis.

Correlations Between Serum Cytokine Levels and the Use of a Moisturizer in Elderly Women in Accordance with the Improvement of Objective and Subjective Skin Condition.

Purpose: In the skin of elderly people with dryness, the production of inflammatory cytokines tends to be induced under the influence of external stimuli. Therefore, there has been a hypothesis that the deterioration of skin conditions due to aging is linked to systemic inflammation. This study aimed to verify the possibility that the use of moisturizer improves skin condition and suppresses systemic inflammation. Methods: As an open study, the participants (n=75) were randomly assigned to either control group or moisturizer group. Participants in the moisturizer group used a moisturizer called Grafa Moisture Keep Milk MC at least twice a day for four weeks on the entire body below the neck. Objective skin conditions (overall dry skin score, water content of the stratum corneum, and transepidermal water loss) and serum cytokine levels (IL-1α, IL-1ß, IL-4, IL-5, IL-6, IL-8, and TNF-α) were evaluated before and after the study in both groups. Subjective skin condition (questionnaire evaluation) was also assessed in the moisturizer group after the study. Results: Serum IL-6 level was significantly reduced in the moisturizer group (n=16) compared with the control group (n=36). In addition, there was an inverse correlation between serum IL-5 and the subjective moisturizing effect in the questionnaire evaluation, suggesting that the moisturizer improved subjective symptoms of dryness by reducing IL-5 levels. Furthermore, there was a positive correlation between IL-5 and IL-6, indicating that they are regulated by common upstream factors. A significant positive correlation of transepidermal water loss with serum IL-4 levels was also detected. Conclusion: The application of the moisturizer to the entire body not only improved subjective and objective skin condition, it may also reduce the levels of circulating inflammatory cytokines. Umin Clinical Trials Registry: Registration number: UMIN 000052024.

Analysis of skin aging patterns using a facial imaging system in patients with atopic dermatitis.

BACKGROUND: There are few studies on skin aging in patients with atopic dermatitis (AD). OBJECTIVES: To clarify the characteristics of facial skin aging in AD patients. MATERIALS & METHODS: Using facial images obtained by a digital imaging system (VISIA evolution), we compared the severity scores for 10 aging signs in 53 women in the AD group and 29 women in the healthy control group, all 35-49 years old. RESULTS: The severity scores for fine lines on the forehead, periorbital wrinkles, nasolabial folds, and texture of the mouth contour were significantly higher in the AD group than in the controls. However, in order to exclude a direct effect of dermatitis at the time of measurement, cases with signs of AD at the evaluation site were excluded from the AD group (defined as the AD [non-lesion] group), revealing no statistical significance between the AD (non-lesion) group and the healthy control group for any of the 10 facial signs. Age subset analysis showed that for individuals in their late 40s, the AD (non-lesion) group exhibited significantly higher scores for crow's feet wrinkle and nasolabial fold compared to the healthy control group. Furthermore, these two scores correlated with one other, suggesting that they may be induced by the same factors. CONCLUSION: The results of this study show that skin aging associated with AD is prominent in areas prone to transient wrinkling by frequent blinking and speaking or facial expressions. Understanding of the need for appropriate AD treatment from a cosmetic perspective may increase patient adherence.

2-O-Octadecylascorbic acid represses RhoGDIß expression and ameliorates DNA damage-induced abnormal spindle orientations.

The appropriate regulation of spindle orientation maintains proper tissue homeostasis and avoids aberrant tissue repair or regeneration. Spindle misorientation due to imbalance or improper functioning leads to a loss of tissue integrity and aberrant growth, such as tissue loss or overgrowth. Pharmacological manipulation to prevent spindle misorientation will enable a better understanding of how spindle orientation is involved in physiological and pathological conditions and will provide therapeutic possibilities to treat patients associated with abnormal tissue function caused by spindle misorientation. N-terminal-deleted Rho guanine nucleotide dissociation inhibitor ß (RhoGDIß/RhoGDI2/LyGDI) produced by caspase-3 activation perturbs spindle orientation in surviving cells following exposure to either ionizing radiation or UVC. Thus, presumably, RhoGDIß cleaved by caspase-3 activation acts as a determinant of radiation-induced spindle misorientation that promote aberrant tissue repair due to deregulation of directional organization of cell population and therefore becomes a potential target of drugs to prevent such response. The objective of this study was to screen and identify chemicals that suppress RhoGDIß expression. We focused our attention on ascorbic acid (AA) derivatives because of their impact on the maintenance of skin tissue homeostasis. Here, we screened for AA derivatives that suppress RhoGDIß expression in HeLa cells and identified a lipophilic derivative, 2-O-octadecylascorbic acid (2-OctadecylAA), as a novel RhoGDIß inhibitor that ameliorated ionizing radiation-induced abnormal spindle orientations. Among all examined AA derivatives, which were also antioxidative, the inhibition activity was specific to 2-OctadecylAA. Therefore, this activity was not due to simple antioxidant properties. 2-OctadecylAA was previously shown to prevent hepatocellular carcinoma development. Our findings suggest that the anticarcinogenic effects of 2-OctadecylAA are partly due to RhoGDIß inhibition mechanisms by which spindle orientation perturbations are attenuated. Thus, the molecular targeting features of RhoGDIß warrant its further development for the treatment or control of spindle orientation abnormalities that affect epithelial homeostasis.

Physiological Skin Characteristics of Infants and Children Compared to Those of Women.

Introduction There is little information regarding skin conditions in infants and children, especially with respect to age, anatomical sites, and seasonal variations. This study aimed to compare the physiological skin characteristics of infants and children with those of women. Methods This study involved skin measurements and a questionnaire-based survey assessing healthy infants and children aged one month to six years and four months (37 males and 48 females) and 15 healthy women in their twenties in the summer, and healthy infants and children aged two months to six years and seven months (34 males and 45 females) and 15 healthy women in their twenties in the winter. The physiological characteristics of the skin of infants and children were surveyed by age. We excluded infants and children with allergic symptoms at the time of measurement. There were 11 subjects with a history of atopic dermatitis. Results Compared with women, infants and children had lower stratum corneum water content and higher transepidermal water loss (TEWL) at most sites. Minimal sebum secretion was observed throughout the body in infants and children aged ≥1 year. The skin surface pH of infants and children was low throughout the body. The questionnaire revealed that skin issues were most common at the anterior neck and cubital fossa, where TEWL was markedly high. These results suggest that barrier function is less developed in the skin of infants and children than in the skin of women. Conclusions The physiological characteristics of skin varied depending on age, anatomical site, and season; hence, skincare guidance must be provided according to these factors.

Investigation of partially myristoylated carboxymethyl chitosan, an amphoteric-amphiphilic chitosan derivative, as a new material for cosmetic and dermal application.

BACKGROUND: Cationic amphiphilic chitosan derivatives can form polymeric micelles, which are useful cosmetic materials, but they form polyion complexes with anionic polymers, which can cause formulation difficulties. AIMS: This study aimed to evaluate the usefulness of partially myristoylated carboxymethyl chitosan, an amphoteric-amphiphilic chitosan derivative, as a new material for cosmetics in the absence of a surfactant comprising an anionic polymer. METHODS: An anionic polymer and 1,2-decanediol (an antimicrobial agent)-containing partially myristoylated carboxymethyl chitosan nanoemulsified lotion and glabridin (an antimelanogenic agent)-containing partially myristoylated carboxymethyl chitosan polymeric micelle were prepared using a pressure homogenization method. The release of interleukin-1α, cell viability, and melanogenesis inhibition was evaluated on a human skin model. Antimicrobial activity was evaluated using agar dilution method. RESULTS: A mixture of partially myristoylated carboxymethyl chitosan and carboxyvinyl polymer did not form a polyion complex, but it formed a hydrophilic gel. The anionic polymer-containing partially myristoylated carboxymethyl chitosan nanoemulsified formulation was stable, with no decrease in cell viability and horny layer exfoliation, which are typically observed with Tween 60. Compared with the formulation with methyl paraben (0.2%), the formulation to which 1,2-decanediol (0.05%) was added improved the antibacterial activity against methicillin-resistant Staphylococcus aureus and Propionibacterium acnes; however, no interleukin-1α upregulation was observed. The glabridin-containing partially myristoylated carboxymethyl chitosan polymeric micelles enhanced melanogenesis inhibition and percutaneous glabridin delivery to the epidermis compared with conventional emulsified micelles. CONCLUSIONS: These results suggest that partially myristoylated carboxymethyl chitosan-forming polymeric micelles, in combination with 1,2-decanediol and glabridin, may be useful for surfactant-free cosmetic emulsions.

Adipose-derived stromal/stem cells improve epidermal homeostasis.

Wound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

RhoGDIß affects HeLa cell spindle orientation following UVC irradiation.

The molecular signals that regulate mitotic spindle orientation to determine the proper division axis play a critical role in the development and maintenance of tissue homeostasis. However, deregulation of signaling events can result in spindle misorientation, which in turn can trigger developmental defects and cancer progression. Little is known about the cellular signaling pathway involved in the misorientation of proliferating cells that evade apoptosis after DNA damage. In this study, we found that perturbations to spindle orientation were induced in ultraviolet C (UVC)-irradiated surviving cells. N-terminal truncated Rho GDP-dissociation inhibitor ß (RhoGDIß), which is produced by UVC irradiation, distorted the spindle orientation of HeLa cells cultured on Matrigel. The short hairpin RNA-mediated knockdown of RhoGDIß significantly attenuated UVC-induced misorientation. Subsequent expression of wild-type RhoGDIß, but not a noncleavable mutant, RhoGDIß (D19A), again led to a relative increase in spindle misorientation in response to UVC. Our findings revealed that RhoGDIß impacts spindle orientation in response to DNA damage.

Efficient Percutaneous Delivery of the Antimelanogenic Agent Glabridin Using Cationic Amphiphilic Chitosan Micelles.

Partially myristoylated chitosan pyrrolidone carboxylate (PMCP) is a cationic amphiphilic chitosan derivative. Glabridin (Glab) from licorice root extracts is a hydrophobic antimelanogenic agent. Here we assessed the effects of cationic Glab-containing polymeric micelles derived from PMCP (Glab/PMCP-PM) on the ability of Glab to penetrate the skin and inhibit melanogenesis using a human skin model. The amount of Glab absorbed 24 h after the application of Glab/PMCP-PM was approximately four times higher than that of conventional oil-in-water micelles (control) prepared using Tween 60. Further, the release of IL-1α, a mediator of inflammation, was not detected. Treatment with Glab/PMCP-PM significantly increased the inhibition of melanogenesis compared with control. The inhibition of melanogenesis depends upon the enhanced ability of Glab to penetrate the skin, particularly the epidermis. Moreover, the inhibition of melanogenesis and the cationic potential of the Glab/PMCP-PM levels were increased by the cationic phospholipid copolymer. Therefore, Glab/PMCP-PM shows potential as an effective transdermal delivery system for treating skin hyperpigmentation.

Radiation-Induced RhoGDIß Cleavage Leads to Perturbation of Cell Polarity: A Possible Link to Cancer Spreading.

The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase-3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase-3. RhoGDIß, known as a direct cleavage substrate of caspase-3, is overexpressed in many epithelial cancers. The N-terminal-truncated RhoGDIß (ΔN-RhoGDIß) is accumulated in caspase-3-activated cells. Stable expression of ΔN-RhoGDIß in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound-healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN-RhoGDIß but not wild-type RhoGDIß was present in the detergent-soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN-RhoGDIß, resulting in increased radiation-induced compensatory proliferation linking to RhoA activation. Thus, ΔN-RhoGDIß dominant-negatively regulates Cdc42 activity and contributes to loss of polarity-related functions. The caspase-3-cleaved RhoGDIß is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493-2505, 2016. © 2016 Wiley Periodicals, Inc.

Cinnamtannin B-1 Promotes Migration of Mesenchymal Stem Cells and Accelerates Wound Healing in Mice.

Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.

Mallotus philippinensis bark extracts promote preferential migration of mesenchymal stem cells and improve wound healing in mice.

In the present study, we report the effects of the ethanol extract from Mallotus philippinensis bark (EMPB) on mesenchymal stem cell (MSC) proliferation, migration, and wound healing in vitro and in a mouse model. Chemotaxis assays demonstrated that EMPB acted an MSC chemoattractant and that the main chemotactic activity of EMPB may be due to the effects of cinnamtannin B-1. Flow cytometric analysis of peripheral blood mononuclear cells in EMPB-injected mice indicated that EMPB enhanced the mobilization of endogenous MSCs into blood circulation. Bioluminescent whole-animal imaging of luciferase-expressing MSCs revealed that EMPB augmented the homing of MSCs to wounds. In addition, the efficacy of EMPB on migration of MSCs was higher than that of other skin cell types, and EMPB treatment improved of wound healing in a diabetic mouse model. The histopathological characteristics demonstrated that the effects of EMPB treatment resembled MSC-induced tissue repair. Taken together, these results suggested that EMPB activated the mobilization and homing of MSCs to wounds and that enhancement of MSC migration may improve wound healing.

Acceleration of UVB-induced photoageing in nrf2 gene-deficient mice.

Ultraviolet (UV) radiation is one of the most important environmental factors involved in the pathogenesis of premature skin ageing, termed photoageing. The harmful effects of UV in photoageing are associated with the generation of reactive oxygen species, and cellular antioxidants act to prevent the occurrence and reduce the severity of UV-induced photoageing. The transcription factor Nrf2 and its cytoplasmic anchor protein, Keap1, are central regulators of the cellular antioxidant response. Here, we investigated the role of the Nrf2-Keap1 pathway in photoageing using nrf2 gene-deficient (nrf2(-/-)) mice. Our results indicated that UVB-irradiated nrf2(-/-) mice showed accelerated photoageing, such as coarse wrinkle formation, loss of skin flexibility, epidermal thickening and deposition of extracellular matrix in the upper dermis. In addition, nrf2(-/-) mice also showed an increase in cutaneous reactivity for the lipid peroxidation product 4-hydroxy-2-nonenal and a significant decrease in cutaneous glutathione level. These findings indicate that Nrf2 plays the important role in the protection against UVB-induced photoageing.

Oxidative stress induces the formation of D-aspartyl residues in the elastin mimic peptides.

Racemization of aspartyl (Asp) residues in peptides and proteins has been considered to be a nonenzymatic chemical reaction which occurs via succinimide formation. However, it has not been known yet what conditions in living body accelerate the inversion of the L- to the D-form. Here, we examined the effect of ultraviolet (UV) exposure or oxidative stress on the formation of D-Asp residues in the elastin mimic peptides with or without heat treatment. As a result, UV exposure did not affect the D-Asp formation in peptides. On the other hand, the amount of D-Asp in heat-treated peptide solution at the same time as addition of HO(.) radical, H(2)O(2), and lipid peroxide was significantly increased. These results indicate that the inversion rate to D-form of Asp residues in skin elastin is accelerated by generation of reactive oxygen species (ROS), and that oxidative stress might be closely related to D-Asp formation in aging proteins.

Hapten-induced contact hypersensitivity is enhanced in Tyk2-deficient mice.

BACKGROUND: Previous studies have shown that Tyk2, a member of the Janus family of protein tyrosine kinases, which are activated by a variety of cytokines, plays a crucial role in interleukin (IL)-12-mediated T-cell functions such as IFN-gamma production. On the other hand, hapten-induced contact hypersensitivity (CHS) is mediated by IFN-gamma producing CD8+ T cells and regulated by CD4+ T cells. OBJECTIVE: This study hypothesized that the CHS response might be reduced in Tyk2-deficient mice because of a lack of IFN-gamma production from CD4+ and CD8+ T cells. METHODS: The CHS reaction was evoked in wild-type and Tyk2-deficient mice and the ears of the mice were examined to measure for several cytokines. RESULTS: Ear swelling during CHS was significantly enhanced in Tyk2-deficient mice compared with the controls. IL-12 and IFN-gamma levels at the reaction sites in Tyk2-deficient mice were significantly lower than in the controls, whereas IL-2 and IL-4 levels were elevated. Furthermore, STAT3- and STAT4-phosphorylation in the draining lymph node cells of Tyk2-deficient mice decreased. CONCLUSION: These results suggest that the lack of Tyk2-mediated signal transduction enhances a compensative pathway during CHS.