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460 Development of an in vitro method to evaluate the skin hydration potential of topical formulations
Epidermal Structure and Barrier Function | ABSTRACTS 456
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Epidermal dysfunction leads to an age-associated increase in levels of serum inflammatory cytokines L Hu1, T Mauro2, E Dang3, G Man2, J Zhang1, D Lee2, G Wang4, K Feingold2, PM Elias2 and M Man2 1 Tianjin Medl Univ, Tianjin, China, 2 VA Med Ctr/UCSF, San Francisco, CA, 3 Xijing Hosp, China and 4 Xijing Hospital, Xi’an, China Elderly populations display chronic inflammation, which is associated with development of age-associated disorders; i.e., type II diabetes and cardiovascular disorders. But the origin of age-associated systemic inflammation is unknown. Our prior studies showed that abnormalities in epidermal function provoke cutaneous inflammation, and that intrinsically -ged mouse and human skin display compromised permeability barrier homeostasis and reduced stratum corneum hydration. Thus, we hypothesized that epidermal dysfunction could contribute to systemic inflammation in the elderly. Our results show first, that acute disruption of the epidermal permeability barrier in young mice leads to a rapid increase in cutaneous cytokine mRNA expression and cytokine levels in serum. Second, cytokine levels increase in skin and serum of aged mice (>12 mos). Third, TNFa and amyloid A mRNA expression increased in epidermis, but not in the liver of aged mice, in parallel with a significant elevation in cytokine serum levels. Fourth, disruption of the permeability barrier induced comparable elevations in epidermal and serum cytokine levels in normal and athymic mice, suggesting that T cells play a negligible role in elevations in cutaneous and serum inflammatory cytokines induced by epidermal dysfunction. Fifth, correction of epidermal function significantly reduced cytokine levels in the skin and serum of aged mice. Together, these results suggest that sustained abnormalities in epidermal function in aged skin contribute to systemic inflammation, potentially predisposing the elderly to the subsequent development or exacerbation of chronic inflammatory disorders.
Production of Serotonin and N-acetylserotonin in the human skin in vivo A Slominski1, T Kim2, I Semak3, T Sweatman4, C Skobowiat5, J Steketee4, Z Lin4, A Postlethwaite6, W Li4 and D Tobin7 1 University of Alabama at Birmingham & VA Medical Center, Birmingham, AL, 2 University of Alabama at Birmingham, Birmingham, AL, 3 Belarusian State University, Minsk, Belarus, 4 University of Tennessee Health Science Center, Memphis, TN, 5 Nicolaus Copernicus University, Bydgoszcz, Poland, 6 University of Tennessee Health Science Center & VA Medical Center, Memphis, TN and 7 University of Bradford, Bradford, United Kingdom Serotonin and its metabolite N-acetylserotonin (NAS) are identifiable by liquid chromatography mass spectrometry (LC/MS) in extracted human epidermis. Endogenous epidermal levels were 113.18 13.34 and 43.41 12.45 ng/mg protein for serotonin and NAS, respectively. Their production was independent of race, gender and age, except for higher NAS levels in young African-Americans [30 to 50 years old (yo)] vs old Caucasian males (60 to 90 yo). NAS was also detected in human serum at a concentration 1.70 0.09 ng/mL, being 174 times lower than that of serotonin. While there were no age- or sex-dependent differences in serum serotonin levels, serum NAS levels were higher in females. Tryptophan hydroxylase (TPH) activity was detected in epidermal melanocytes and fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 mmol/min. Low but detectable TPH activity was seen in cultured epidermal keratinocytes. Serotonin was also produced endogenously in cultured melanocytes, keratinocytes and dermal fibroblasts, with UVB modulating this production. Immnocytochemistry localized serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells and dermal fibrocytes. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal and adnexal compartments of human skin and in cultured skin cells, and NAS is detectable in human serum.
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Cellular and metabolic basis for the ichthyotic phenotype in ichthyin deficiency E Mauldin1, ML Cassal1, S Jeong2, K Vavrova3, Y Uchida4, K Park4, B Craiglow5, K Choate5, KO Shin6, Y Lee7, D Khnykin8, G Grove9 and PM Elias4 1 U Penn, Philadelphia, PA, 2 Seowon Univ, Daejeon, Republic of Korea, 3 Charles Univ, Prague, Czech Republic, 4 VA Med Ctr/UCSF, San Francisco, CA, 5 Yale Sch of Med, New Haven, CT, 6 Chungbuk Natl Univ, Cheongju, Republic of Korea, 7 Chungbuk Natl Univ, Cheongju, Republic of Korea, 8 Oslo Univ Hosp, Oslo, Norway and 9 cyberDERM, Broomall, PA Abnormal barrier in the ichthyoses ‘drives’ the clinical phenotype via a ‘best attempt’ to form a barrier sufficient for terrestrial life. Mutations in several lipid synthetic enzymes produce autosomal recessive congenital ichthyosis (ARCI), with blockade of fatty acid (FA) and ceramide (Cer) production, critical for barrier formation. Mutations in ichthyin (NIPAL4) compromise FACoA esterification, because NIPAL4 encodes a protein that delivers Mg++, required for the acylCoA synthetase function of FA transport protein 4 (FATP4), mutated in ichthyosis prematurity syndrome. Barrier function was abnormal in both patients and 4 American bulldogs with NIPAL4 mutations. The barrier abnormality may be attributed to: i) wide-spread cytotoxicity, manifested as membrane stripping, likely due to detergent activity of non-esterified free FAs compromising lamellar body secretion; and ii) failure to form the corneocyte lipid envelope (CLE), the scaffold for formation of the lamellar bilayers. CLE restoration with topical acylCer normalized it, but the visible phenotype and barrier abnormality only partially improved, due to persistent cytotoxicity. These results illuminate functional, cellular and metabolic contributors to the phenotype in ichthyin deficiency. Analogous with a disorder of distal cholesterol metabolism (CHILD syndrome), provision of pathway end-product alone does not suffice to correct the clinical phenotype. Ichthyin deficiency represents another lipid synthetic ARCI that converges on the CLE, compromising its scaffold function.
Development of an in vitro method to evaluate the skin hydration potential of topical formulations M Bachelor, K Guntur, A Plotkin, J Oldach and A Armento MatTek Corporation, Ashland, MA Skin hydration is vital in maintaining the barrier function of skin. Moisturizers, including creams, gels and lotions contain substances that both promote retention of water and reduce water loss from the skin. Current moisturizer product development is limited to expensive and highly variable human clinical studies and cell based models which lack relevant tissue structure and function. In addition, cell based models restrict potential test compounds/formulations due to solubility issues. In this study, EpiDermFT, an in vitro 3D human skin model, was used to evaluate the skin hydration potential of several commercially available skin moisturizers. EpiDermFT is cultured at the Air-liquid Interface and is amenable to topical exposures of test compounds or formulations regardless of water solubility. Following a 90 minute equilibration at room temperature and humidity, the in vitro skin model was exposed to various skin moisturizers for 60 minutes. Following exposure, the moisturizers were removed and skin hydration was immediately evaluated by measuring the electrical impedance of the tissue surface (DPM 9003 Nova Meter, Nova Technologies). Total RNA was extracted from the epidermis and changes in the expression of genes modulating barrier function were evaluated. Analysis of skin hydration by electrical impedance demonstrated significant 2-5 fold differences between moisturizer treated tissues and untreated or PBS treated tissues.. Furthermore, although all moisturizers demonstrated increased epidermal hydration, significant differences were observed among the various moisturizers tested. Altogether, assessment of skin hydration following treatment with topically applied moisturizers in the EpiDermFT in vitro human skin model is a valuable in vitro method for use in product development and claims substantiation studies.
Evaluation of intermittent treatment with cosmetic compounds on skin J Idkowiak-Baldys, F Liebel and J Lyga AVON Products, Inc., Suffern, NY Continuous, topical treatment with biologically active ingredients such as retinol or glycolic acid has the potential of delivering demonstrable antiaging benefits to skin. In some instances, however, especially for sensitive skin consumers, those benefits can be overshadowed by undesirable attributes such as irritation. In these instances, treatments may be paused then resumed after a brief respite. To understand the impact of this practice, the efficacy of several active ingredients was evaluated in a continuous vs. intermittent treatment. Retinol, glycolic acid, and phytol were used to treat human dermal fibroblasts in vitro. The effect of the compounds on production of extracellular matrix components was evaluated after continuous treatment (fresh media and treatment added every other day for 12 days) vs. intermittent manner (2 days of active treatments alternated with 2 days without active for total of 12 days). Two of the ingredients tested (retinol and phytol) showed an enhanced effect on hyaluronic acid production, a biomarker associated with skin aging, when applied in an intermittent vs. continuous manner. To evaluate if this in vitro response translates, various doses of retinol were tested in a continuous vs. intermittent treatment in vivo. Retinol was applied to upper inner arm of subjects daily for three weeks or for three days followed by 4 days of no treatment for three weeks. Effects on the skin were measured non-invasively looking at skin redness, barrier function, cell turnover and epidermal thickening. Initial findings are presented related to the potential of this approach to deliver more tolerable efficacious anti-aging treatments.
Recent evolution for an involucrin/sperm mitochondria haplotype associated with epidermal function in modern humans Z Goodwin1, I Oh1, M Mathyer2, A Quiggle2 and C de Guzman Strong2 1 Washington University School of Medicine, Saint Louis, MO and 2 Washington University in St Louis, St Louis, MO The Epidermal Differentiation Complex (EDC) was identified as the most rapidly evolving locus in the human genome. We sought to determine the EDC genetic variants undergoing positive selection across modern human populations. Causal variants in positively selected regions were obtained using CMS scores for each of the representative EDC genes in 3 geographically distinct HapMap populations (CEU, YRI, CHB). The highest CMS-scores were found in 3 SNPs in the IVL gene and represent a haplotype in the CEU (European) population (MAF, 95%). Closer investigation of the allele frequencies of the IVL haplotype with respect to latitude in 13 populations identified a strong and direct correlation (r2¼0.88) for selection of the IVL haplotype with northern latitudes suggesting a fitness advantage for the allele in these regions. GTEx data revealed a dose-dependent and skin-specific eQTL with the IVL haplotype suggesting a compelling argument for an epigenetic variant for positive selection for the IVL haplotype. Luciferase reporter assays for rs48458327 in the nearby 923 enhancer in strong LD with the IVL haplotype revealed a significant increase in enhancer activity in support of an enhancer allele underlying IVL haplotype positive selection. CRISPR/Cas9-mediated deletion of the 923 enhancer in mice demonstrated a requirement for 923 in the cis regulation of nearby Ivl and Lce6a expression as well as Smcp, a sperm mitochondrial cysteine protein. Reduced progeny were observed from 923-/- male consistent with male sperm motility defects in Smcp-/- mice. Together, our results identify human positive selection for an IVL haplotype associated with a fitness advantage in northern latitudes that could likely be explained by a 923 enhancer variant affecting both Ivl, Lce6a, and Smcp expression in mice.
www.jidonline.org S79
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Epidermal dysfunction leads to an age-associated increase in levels of serum inflammatory cytokines L Hu1, T Mauro2, E Dang3, G Man2, J Zhang1, D Lee2, G Wang4, K Feingold2, PM Elias2 and M Man2 1 Tianjin Medl Univ, Tianjin, China, 2 VA Med Ctr/UCSF, San Francisco, CA, 3 Xijing Hosp, China and 4 Xijing Hospital, Xi’an, China Elderly populations display chronic inflammation, which is associated with development of age-associated disorders; i.e., type II diabetes and cardiovascular disorders. But the origin of age-associated systemic inflammation is unknown. Our prior studies showed that abnormalities in epidermal function provoke cutaneous inflammation, and that intrinsically -ged mouse and human skin display compromised permeability barrier homeostasis and reduced stratum corneum hydration. Thus, we hypothesized that epidermal dysfunction could contribute to systemic inflammation in the elderly. Our results show first, that acute disruption of the epidermal permeability barrier in young mice leads to a rapid increase in cutaneous cytokine mRNA expression and cytokine levels in serum. Second, cytokine levels increase in skin and serum of aged mice (>12 mos). Third, TNFa and amyloid A mRNA expression increased in epidermis, but not in the liver of aged mice, in parallel with a significant elevation in cytokine serum levels. Fourth, disruption of the permeability barrier induced comparable elevations in epidermal and serum cytokine levels in normal and athymic mice, suggesting that T cells play a negligible role in elevations in cutaneous and serum inflammatory cytokines induced by epidermal dysfunction. Fifth, correction of epidermal function significantly reduced cytokine levels in the skin and serum of aged mice. Together, these results suggest that sustained abnormalities in epidermal function in aged skin contribute to systemic inflammation, potentially predisposing the elderly to the subsequent development or exacerbation of chronic inflammatory disorders.
Production of Serotonin and N-acetylserotonin in the human skin in vivo A Slominski1, T Kim2, I Semak3, T Sweatman4, C Skobowiat5, J Steketee4, Z Lin4, A Postlethwaite6, W Li4 and D Tobin7 1 University of Alabama at Birmingham & VA Medical Center, Birmingham, AL, 2 University of Alabama at Birmingham, Birmingham, AL, 3 Belarusian State University, Minsk, Belarus, 4 University of Tennessee Health Science Center, Memphis, TN, 5 Nicolaus Copernicus University, Bydgoszcz, Poland, 6 University of Tennessee Health Science Center & VA Medical Center, Memphis, TN and 7 University of Bradford, Bradford, United Kingdom Serotonin and its metabolite N-acetylserotonin (NAS) are identifiable by liquid chromatography mass spectrometry (LC/MS) in extracted human epidermis. Endogenous epidermal levels were 113.18 13.34 and 43.41 12.45 ng/mg protein for serotonin and NAS, respectively. Their production was independent of race, gender and age, except for higher NAS levels in young African-Americans [30 to 50 years old (yo)] vs old Caucasian males (60 to 90 yo). NAS was also detected in human serum at a concentration 1.70 0.09 ng/mL, being 174 times lower than that of serotonin. While there were no age- or sex-dependent differences in serum serotonin levels, serum NAS levels were higher in females. Tryptophan hydroxylase (TPH) activity was detected in epidermal melanocytes and fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 mmol/min. Low but detectable TPH activity was seen in cultured epidermal keratinocytes. Serotonin was also produced endogenously in cultured melanocytes, keratinocytes and dermal fibroblasts, with UVB modulating this production. Immnocytochemistry localized serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells and dermal fibrocytes. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal and adnexal compartments of human skin and in cultured skin cells, and NAS is detectable in human serum.
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Cellular and metabolic basis for the ichthyotic phenotype in ichthyin deficiency E Mauldin1, ML Cassal1, S Jeong2, K Vavrova3, Y Uchida4, K Park4, B Craiglow5, K Choate5, KO Shin6, Y Lee7, D Khnykin8, G Grove9 and PM Elias4 1 U Penn, Philadelphia, PA, 2 Seowon Univ, Daejeon, Republic of Korea, 3 Charles Univ, Prague, Czech Republic, 4 VA Med Ctr/UCSF, San Francisco, CA, 5 Yale Sch of Med, New Haven, CT, 6 Chungbuk Natl Univ, Cheongju, Republic of Korea, 7 Chungbuk Natl Univ, Cheongju, Republic of Korea, 8 Oslo Univ Hosp, Oslo, Norway and 9 cyberDERM, Broomall, PA Abnormal barrier in the ichthyoses ‘drives’ the clinical phenotype via a ‘best attempt’ to form a barrier sufficient for terrestrial life. Mutations in several lipid synthetic enzymes produce autosomal recessive congenital ichthyosis (ARCI), with blockade of fatty acid (FA) and ceramide (Cer) production, critical for barrier formation. Mutations in ichthyin (NIPAL4) compromise FACoA esterification, because NIPAL4 encodes a protein that delivers Mg++, required for the acylCoA synthetase function of FA transport protein 4 (FATP4), mutated in ichthyosis prematurity syndrome. Barrier function was abnormal in both patients and 4 American bulldogs with NIPAL4 mutations. The barrier abnormality may be attributed to: i) wide-spread cytotoxicity, manifested as membrane stripping, likely due to detergent activity of non-esterified free FAs compromising lamellar body secretion; and ii) failure to form the corneocyte lipid envelope (CLE), the scaffold for formation of the lamellar bilayers. CLE restoration with topical acylCer normalized it, but the visible phenotype and barrier abnormality only partially improved, due to persistent cytotoxicity. These results illuminate functional, cellular and metabolic contributors to the phenotype in ichthyin deficiency. Analogous with a disorder of distal cholesterol metabolism (CHILD syndrome), provision of pathway end-product alone does not suffice to correct the clinical phenotype. Ichthyin deficiency represents another lipid synthetic ARCI that converges on the CLE, compromising its scaffold function.
Development of an in vitro method to evaluate the skin hydration potential of topical formulations M Bachelor, K Guntur, A Plotkin, J Oldach and A Armento MatTek Corporation, Ashland, MA Skin hydration is vital in maintaining the barrier function of skin. Moisturizers, including creams, gels and lotions contain substances that both promote retention of water and reduce water loss from the skin. Current moisturizer product development is limited to expensive and highly variable human clinical studies and cell based models which lack relevant tissue structure and function. In addition, cell based models restrict potential test compounds/formulations due to solubility issues. In this study, EpiDermFT, an in vitro 3D human skin model, was used to evaluate the skin hydration potential of several commercially available skin moisturizers. EpiDermFT is cultured at the Air-liquid Interface and is amenable to topical exposures of test compounds or formulations regardless of water solubility. Following a 90 minute equilibration at room temperature and humidity, the in vitro skin model was exposed to various skin moisturizers for 60 minutes. Following exposure, the moisturizers were removed and skin hydration was immediately evaluated by measuring the electrical impedance of the tissue surface (DPM 9003 Nova Meter, Nova Technologies). Total RNA was extracted from the epidermis and changes in the expression of genes modulating barrier function were evaluated. Analysis of skin hydration by electrical impedance demonstrated significant 2-5 fold differences between moisturizer treated tissues and untreated or PBS treated tissues.. Furthermore, although all moisturizers demonstrated increased epidermal hydration, significant differences were observed among the various moisturizers tested. Altogether, assessment of skin hydration following treatment with topically applied moisturizers in the EpiDermFT in vitro human skin model is a valuable in vitro method for use in product development and claims substantiation studies.
Evaluation of intermittent treatment with cosmetic compounds on skin J Idkowiak-Baldys, F Liebel and J Lyga AVON Products, Inc., Suffern, NY Continuous, topical treatment with biologically active ingredients such as retinol or glycolic acid has the potential of delivering demonstrable antiaging benefits to skin. In some instances, however, especially for sensitive skin consumers, those benefits can be overshadowed by undesirable attributes such as irritation. In these instances, treatments may be paused then resumed after a brief respite. To understand the impact of this practice, the efficacy of several active ingredients was evaluated in a continuous vs. intermittent treatment. Retinol, glycolic acid, and phytol were used to treat human dermal fibroblasts in vitro. The effect of the compounds on production of extracellular matrix components was evaluated after continuous treatment (fresh media and treatment added every other day for 12 days) vs. intermittent manner (2 days of active treatments alternated with 2 days without active for total of 12 days). Two of the ingredients tested (retinol and phytol) showed an enhanced effect on hyaluronic acid production, a biomarker associated with skin aging, when applied in an intermittent vs. continuous manner. To evaluate if this in vitro response translates, various doses of retinol were tested in a continuous vs. intermittent treatment in vivo. Retinol was applied to upper inner arm of subjects daily for three weeks or for three days followed by 4 days of no treatment for three weeks. Effects on the skin were measured non-invasively looking at skin redness, barrier function, cell turnover and epidermal thickening. Initial findings are presented related to the potential of this approach to deliver more tolerable efficacious anti-aging treatments.
Recent evolution for an involucrin/sperm mitochondria haplotype associated with epidermal function in modern humans Z Goodwin1, I Oh1, M Mathyer2, A Quiggle2 and C de Guzman Strong2 1 Washington University School of Medicine, Saint Louis, MO and 2 Washington University in St Louis, St Louis, MO The Epidermal Differentiation Complex (EDC) was identified as the most rapidly evolving locus in the human genome. We sought to determine the EDC genetic variants undergoing positive selection across modern human populations. Causal variants in positively selected regions were obtained using CMS scores for each of the representative EDC genes in 3 geographically distinct HapMap populations (CEU, YRI, CHB). The highest CMS-scores were found in 3 SNPs in the IVL gene and represent a haplotype in the CEU (European) population (MAF, 95%). Closer investigation of the allele frequencies of the IVL haplotype with respect to latitude in 13 populations identified a strong and direct correlation (r2¼0.88) for selection of the IVL haplotype with northern latitudes suggesting a fitness advantage for the allele in these regions. GTEx data revealed a dose-dependent and skin-specific eQTL with the IVL haplotype suggesting a compelling argument for an epigenetic variant for positive selection for the IVL haplotype. Luciferase reporter assays for rs48458327 in the nearby 923 enhancer in strong LD with the IVL haplotype revealed a significant increase in enhancer activity in support of an enhancer allele underlying IVL haplotype positive selection. CRISPR/Cas9-mediated deletion of the 923 enhancer in mice demonstrated a requirement for 923 in the cis regulation of nearby Ivl and Lce6a expression as well as Smcp, a sperm mitochondrial cysteine protein. Reduced progeny were observed from 923-/- male consistent with male sperm motility defects in Smcp-/- mice. Together, our results identify human positive selection for an IVL haplotype associated with a fitness advantage in northern latitudes that could likely be explained by a 923 enhancer variant affecting both Ivl, Lce6a, and Smcp expression in mice.
www.jidonline.org S79