Role of interleukin-6 and endothelin-1 receptors in enhanced melanocyte dendricity of facial spots and suppression of their ligands by niacinamide and tranexamic acid.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities with a hallmark characteristic of increased melanocyte dendricity. OBJECTIVES: To determine (1) potential receptors and/or cytokines that are involved in increased melanocyte dendricity in multiple facial spot types; (2) treatment effects of skin-lightening compounds on identified cytokine release from keratinocytes and on dendricity in melanocytes. METHODS: Facial spots (melasma, solar lentigo, acne-induced post-inflammatory hyperpigmentation) and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). The epidermal supra and basal layers were laser dissected to enrich keratinocyte or melanocyte biology respectively for transcriptome analysis. Melanocyte dendricity was assessed histologically by immunofluorescent staining. Effect of interleukin-6 (IL-6) and endothelin-1 (ET-1) on melanocyte dendricity and melanosome transfer were assessed in human melanocytes or melanocyte-keratinocyte co-culture models. Treatment effects of skin-lightening compounds (niacinamide, tranexamic acid [TxA], sucrose laurate/dilaurate mixture [SDL]) were assessed on IL-6 or ET-1 release from keratinocytes and on dendricity in melanocytes. RESULTS: Transcriptome analysis revealed IL-6 receptor and ET-1 receptor were significantly upregulated compared to the adjacent normal skin, visually confirmed at the protein level through immunostaining. Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin. The addition of IL-6 and ET-1 to cell culture models increased melanocyte dendricity and melanosome transfer. IL-6 release was significantly suppressed by niacinamide and its combination, while ET-1 release was significantly reduced by both niacinamide and TxA. In contrast, SDL acted directly upon melanocytes to reduce dendricity. CONCLUSION: Interleukin-6 and ET-1 receptors are significantly upregulated in multiple facial spot types. The in vitro testing demonstrated their respective ligands increased melanocyte dendricity. Tested skin-lightening compounds showed reduction in release of IL-6/ET-1 from epidermal keratinocytes and/or inhibition of melanocyte dendricity. This work sheds light on pathophysiological mechanism of facial spots and potential new mechanisms of these skin-lightening compounds which warrant further human clinical validation.

Morphological and transcriptional evaluation of multiple facial cutaneous hyperpigmented spots.

Background: Morphological characteristics of major facial hyperpigmented spots have been well documented. However, detailed alterations of respective transcriptional profile for each spot and in-depth comparisons across multiple spot types have not been reported. Objectives: To comprehensively assess and compare multiple facial hyperpigmented spot types at the morphological and molecular levels by utilising transcriptional expression profiling with correlation to quantified histological features. Methods: Multiple types of facial spot biopsies were collected from Chinese women and compared to additional biopsies taken from adjacent healthy skin. The types of spots included Solar Lentigos with both elongated dermal-epidermal junction (DEJ) (SL[E]) and flat DEJ (SL[F]), Seborrhoeic Keratosis (SK), Melasma, Freckles, Post-inflammatory hyperpigmentation of resolving acne (PIH[A]) and other stimuli (PIH[O]). Combined histomorphometry, immunohistology, and transcriptome analysis for suprabasal-epidermis, basal-epidermis, and dermal compartments dissected by Laser Capture Microdissection (LCM) were conducted and compared across different spot types. Results: Each spot type was confirmed to have the unique histological pathology already documented elsewhere. Most of the spot types except Melasma and PIH (A) revealed similar melanocyte density to adjacent skin. All spots exhibited increased melanin synthesis, melanosome transportation, as well as enhanced melanocyte dendricity, however, each spot revealed a distinct transcriptome regulation pattern in pigmentation pathways. Upregulation of pigmentation genes was also observed in the dermis of SL(F), SL(E), SK and PIH(O), associated with significant modulation of DEJ related genes in basal-epidermis and/or dermal compartments, suggesting potential melanocyte infiltration into the dermis due to impaired DEJ quality. Beyond upregulated pigmentation, for most spots, gene expression in the suprabasal-epidermis regulating keratinisation was significantly upregulated in conjunction with thickened stratum corneum. Furthermore, downregulation of tight junction related genes represented by claudin-1 was observed in majority of spot types, suggesting compromised barrier function could be a similarity across spots. Additionally, Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) was upregulated in all types of spots, indicating involvement of cell senescence as a common theme. Conclusion: This comprehensive and comparative study based on the histological and transcriptional analysis of three skin compartments provided unique insights into specific causations as well as differences and similarities across multiple hyperpigmented spot types.

The combination of sucrose dilaurate and sucrose laurate suppresses HMGB1: an enhancer of melanocyte dendricity and melanosome transfer to keratinocytes.

BACKGROUND: Hyperpigmented spots are common issues in all ethnicities, involving multiple intrinsic and extrinsic factors such as UVB exposure, hormone balance, inflammatory status and ageing. OBJECTIVES: To determine (i) melanocyte dendricity in multiple facial spot types, (ii) impact of High Mobility Group Box 1 (HMGB1), and the combination of sucrose dilaurate and sucrose laurate (SDL) on melanogenesis and melanocyte dendricity, and (iii) SDL effect on facial spots in a human use test. METHODS: Facial spot and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). Histological assessment of melanocyte dendricity was performed for 3 spot types (solar lentigo, melasma and postinflammatory hyperpigmentation) by immunofluorescent staining for c-kit/MITF. Keratinocyte, melanocyte and melanocyte-keratinocyte co-culture models were used to assess HMGB1 release by UVB radiation, the effects of HMGB1 and SDL on melanin production, melanocyte dendricity and melanosome transfer. The effect of an SDL-containing moisturizer on appearance of facial hyperpigmented spots was assessed against a vehicle control in an 8-week human use test. RESULTS: Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin across three investigated spot types. In cell culture models, a moderate UVB-radiation exposure caused release of HMGB1 from keratinocytes. HMGB1 did not alter melanin production in melanocytes, but enhanced melanocyte dendricity and melanosome transfer. SDL reduced HMGB1 release from keratinocytes, inhibited melanin production, reversibly suppressed melanocyte dendricity and reduced melanosome transfer. In the human use test, SDL-containing moisturizer reduced appearance of spots versus vehicle. CONCLUSION: Increased melanocyte dendricity was observed in multiple types of facial spots. Addition of HMGB1 protein increased melanocyte dendricity and melanosome transfer in cell cultures, implicating potential involvement in spot formation. SDL suppressed melanin production, melanocyte dendricity and melanosome transfer in vitro and reduced appearance of spots in the use test, suggesting SDL is an effective solution to address hyperpigmented spot concerns.

Optimized low pH formulation of niacinamide enhances induction of autophagy marker ATG5 gene expression and protein levels in human epidermal keratinocytes.

BACKGROUND: Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin. OBJECTIVE: To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models. METHODS: Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed. RESULTS: Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48 h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6 h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8. CONCLUSIONS: We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction.

Autophagy activators stimulate the removal of advanced glycation end products in human keratinocytes.

BACKGROUND: The accumulation of advanced glycation end products (AGEs) can impact cellular homoeostasis and protein structure, thus is implicated in numerous skin conditions including yellow, dull appearance. AGE formation is irreversible; thus, understanding of the recycling process of AGEs in the skin is critical for addressing skin appearance conditions. OBJECTIVE: To determine whether (i) accumulation of AGEs occurs in dull appearance group among young population (age 20-29) (ii) in vitro autophagy stimulation results in reduction of AGEs in keratinocytes. METHODS: Facial cheek biopsies were collected from Chinese women (age 20-50) exhibiting either dull or non-dull appearing skin. Histological assessment of glycation was performed for representative subjects among the 20-29 years sub-group by immunofluorescence staining of AGEs. LC-MS methods and keratinocyte cell culture were used to assess impact of autophagy modulators and skin care materials on carboxymethyl lysine (CML) amount, a representative AGE. RESULTS: Notable amounts of AGEs were observed in the epidermal samples among young females. Interestingly, the amount of AGEs was significantly higher among the dull skin appearance group. Treatment of keratinocytes with glyceraldehyde (GLA) enhanced CML in the cells, and postglycation treatment with autophagy activators reduced CML. Two skin care materials, Nymphaea alba flower extract (a.k.a. white water lily extract) and sucrose dilaurate, were identified based from in vitro autophagy activation and found to reduce CML in keratinocytes. CONCLUSION: We found AGEs accumulate in the facial epidermis even among young people, correlating to a yellow and dull appearance. We also demonstrated in vitro activation of autophagy can reduce AGEs in keratinocytes, and autophagy activating skin care materials, N. alba flower extract and sucrose dilaurate, also reduce AGEs in the keratinocyte in vitro model. These data suggest epidermal AGEs contribute to the dull skin appearance, and autophagy activators may provide an effective solution to improve dull appearance by removing and recycling the accumulated glycation in the skin.

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

BACKGROUND: 2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. OBJECTIVES: To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. METHODS: 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. RESULTS: In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. CONCLUSIONS: These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation.

Application of genomics to breakthroughs in the cosmetic treatment of skin ageing and discoloration.

The use of global gene expression profiling, also known as transcriptomics or genomics, provides a means to identify key pathways affected in ageing skin that can be improved with appropriate cosmetic compounds. Aspects of skin ageing that can be addressed include matrix production, barrier, lipid synthesis, antioxidant capacity and hyperpigmentation. Gene expression profiling together with in vitro human skin cell cultures for compound screening and verification have led to the identification of cosmetic compounds and an understanding of the biological effects of compounds such as niacinamide, Pal-KTTKS, hexamidine, retinyl propionate and sodium dehydroacetate. In addition, understanding of the decreased antioxidant capacity of aged skin has led to the identification of new antiageing ingredients, olive-derived fatty acid ethoxylates, which have been shown to restore antioxidant enzymes in skin keratinocytes and fibroblasts. Gene expression profiling of age spots has also provided an understanding of the role of undecylenoyl phenylalanine in reducing melanin production by an adrenergic receptor mechanism in melanocytes. The use of these compounds in cosmetic formulations for skin care can aid improvements in the appearance of aged skin, including the improved appearance of fine lines, wrinkles and age spots.

The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer.

BACKGROUND: Cutaneous hyperpigmentation occurs in multiple conditions. In addition, many Asian women desire a lighter skin colour. Thus, there is a need for the development of skin lightening agents. Niacinamide is a possible candidate. OBJECTIVES: To investigate the effects of niacinamide on melanogenesis in vitro and on facial hyperpigmentation and skin colour in vivo in Japanese women. METHODS: Melanin production was measured in a purified mushroom tyrosinase assay, cultured melanocytes, a keratinocyte/melanocyte coculture model, and a pigmented reconstructed epidermis (PREP) model. The clinical trials included 18 subjects with hyperpigmentation who used 5% niacinamide moisturizer and vehicle moisturizer in a paired design, and 120 subjects with facial tanning who were assigned to two of three treatments: vehicle, sunscreen and 2% niacinamide + sunscreen. Changes in facial hyperpigmentation and skin colour were objectively quantified by computer analysis and visual grading of high-resolution digital images of the face. RESULTS: Niacinamide had no effect on the catalytic activity of mushroom tyrosinase or on melanogenesis in cultured melanocytes. However, niacinamide gave 35-68% inhibition of melanosome transfer in the coculture model and reduced cutaneous pigmentation in the PREP model. In the clinical studies, niacinamide significantly decreased hyperpigmentation and increased skin lightness compared with vehicle alone after 4 weeks of use. CONCLUSIONS: The data suggest niacinamide is an effective skin lightening compound that works by inhibiting melanosome transfer from melanocytes to keratinocytes.