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266 Growth of the cerebriform connective tissue nevus in individuals with Proteus syndrome
Clinical Research II: Pathophysiology and Therapeutics | ABSTRACTS 264
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Topical treatments improved periorbital aging J Namkoong, B Cook, D Kern and HE Knaggs Global R&D, Nu Skin Enterprises, Inc., Provo, UT Facial aging is manifested by different symptoms. One area people pay more attention to and use additional cosmetic products to keep healthier is the eye area e the periorbital region. There are multiple reasons why the periorbital region manifests an unhealthy and aged look. The skin in the eye area is thinner than other parts of face, and repetitive muscle movements and UV exposures lead to skin aging. Periorbital aging includes eyelid drooping, dark circles, under eye bags, or fine lines/wrinkles. Topical and mechanical treatments to improve periorbital aging is challenging due to the proximity to eyes. Hypersensitivity to treatment options are common occurrence in the eyes. In order to improve the skin in the eye area, topical ingredients were evaluated for their potential to modulate different skin aging target genes, such as improving skin structures, hydration, anti-inflammation, antioxidant capacity and cell turnover, using normal human skin equivalent cultures. Some interesting finding includes modulation of Prostaglandin E2 (PGE2). Induction of cellular stress with 5mM or 10mM hydrogen peroxide increased PGE2 expression, which would increase inflammation. In addition, PGE2 increase has been suggested in the skin of elderly and inhibition of PGE2 resulted in collagen production in skin organ culture. Experimental ingredients were able to suppress that H2O2-induced PGE2 expression, similar to ascorbic acid. After evaluating ingredients using in vitro models, a specific formula was developed to target clinical signs of periorbital skin aging. Prior to running a clinical study, the formula was evaluated for safety, due to potential hypersensitivity. This eye formulation did not trigger any safety concerns during in vitro evaluations and non-ocular evaluations. The formula was further assessed for safety during the efficacy testing by both dermatological and ophthalmologic evaluations. This formula improved periorbital aging in 50% of subjects with sagging skin, eyelid firmness and puffiness after 12 weeks of application by dermatological evaluations.
Pilot trial to evaluate the effect of vitamin D on melanocyte biomarkers E Anderson1, M Rezaee2, I Bailey-Healy2, K Sarin2 and J Tang2,1 1 School of Medicine, Stanford University, Stanford, CA and 2 Dermatology, Stanford University, Stanford, CA The role of vitamin D for cancer prevention is an expanding area of interest. In clinical and epidemiologic studies, vitamin D has been shown to predict incidence and severity of melanoma skin cancer. Although the role of vitamin D in melanocyte differentiation and melanoma carcinogenesis has been described, a clear mechanism is yet to be established. Previous studies have suggested that vitamin D supplementation and increased serum vitamin D levels are associated with decreased melanoma incidence. We sought to determine the effect of oral vitamin D supplementation on gene expression within melanocytic nevi of women previously diagnosed with non-melanoma skin cancer. A total of 24 healthy women were included in this randomized placebo-controlled study. Half of the cohort received 4,000 IU oral vitamin D3 daily for 8 weeks. Skin checks were performed at baseline and 8 week follow-up, and nevi were biopsied at both time points. RNA was collected from these nevi for gene expression profiling to identify activity in signaling pathways associated with vitamin D treatment. Women who received vitamin D supplementation experienced a significant increase in vitamin D levels at follow-up (p ¼ 0.0001) while women who received placebo did not (p ¼ 0.50). Gene expression analysis of melanocytic nevi revealed 1048 genes that were significantly differentially expressed between vitamin D-treated and placebo-controlled women. Ingenuity Pathway Analysis indicated that the most significantly differentiated molecular signaling pathways were related to immune system signaling, including primary immunodeficiency signaling (p ¼ 3.53 x 10-6), T cell receptor signaling (p ¼ 4.31 x 10-5), iCOS-iCOSL signaling in T helper cells (p ¼ 1.24 x 10-4), calcium-induced T lymphocyte apoptosis (p ¼ 7.52 x 10-4), and crosstalk between dendritic cells and natural killer cells (p ¼ 1.68 x 10-3). This finding suggests that increased immune surveillance could underlie the previously described association between vitamin D supplementation and decreased melanoma incidence.
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Growth of the cerebriform connective tissue nevus in individuals with Proteus syndrome N Nathan1, R Patel2, M Crenshaw2, M Lindhurst2, C Olsen3, L Biesecker2, K Keppler-Noreuil2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Medical Genomics and Metabolic Genetics Branch, NHGRI, Bethesda, MD and 3 Preventative Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD The plantar cerebriform connective tissue nevus (CCTN) is the most common and problematic cutaneous manifestation of Proteus syndrome, an overgrowth syndrome caused by mosaicism for an activating mutation in AKT1. In order to gain insights into CCTN pathogenesis and natural history, we measured the size of plantar CCTNs using serial images from patients evaluated at the National Institutes of Health Clinical Center. The area of CCTN relative to total plantar area, excluding toes, were measured on 152 images from 22 patients (median age 6.9 years). The relative area of CCTN increased with age in children (n¼17, p<.0001) but not adults. Confluent small papules and nodules extending beyond the boundaries of CCTNs were gradually replaced by fully developed CCTN over time. The center of CCTN overlap from 14 patients appeared to be near the ball of the foot. This unexpected non-random distribution in a mosaic disorder may suggest that growth is augmented by minor trauma from walking. In 8 patients who underwent plantar CCTN biopsy and AKT1 mutational analysis, a positive relationship between CCTN growth rate and mutant allele frequency was observed (Spearman’s rho¼.62, p¼.10). Overall, these observations suggest that the progressive growth of CCTNs is impacted by multiple factors, including age, minor trauma, and the regional density of cells bearing AKT1 mutations.
Once weekly topical all-trans retinol restores type I collagen synthesis in photoaged forearm skin within 4 weeks: A protocol for evaluating anti-aging topical agents P Wang, M Sun, Y Xu, Y Xu, JJ Voorhees, G Fisher and Y Li Dermatology, University of Michigan, Ann Arbor, USA Minor Outlying Islands Type I collagen (COL1) is the major structural protein of human skin. COL1 content declines during aging, leading to skin thinning and fragility in the elderly. This collagen deficiency results in part from reduced COL1 synthesis manifested as reduced COL1 mRNA and type I procollagen (proCOL1) protein levels in aged skin. Daily use of topical retinoids, including all-trans retinol (ROL), has been shown to restore COL1 synthesis in the skin of the elderly. Typically, these studies have involved several months of daily treatment. How fast and to what extent topical ROL can restore COL1 synthesis in photoaged skin to that of young skin has not been well demonstrated. Herein, we investigated a short-term ROL treatment regimen. A oneday occlusion of ROL (0.4%) or vehicle was applied on photoaged forearms of elderly (>65 years old) subjects once a week for 4 weeks. Vehicle was also applied on forearms of young (23-33 years) subjects in the same manner. Skin samples were obtained one week post the last treatment and analyzed for COL1 levels. ROL treatment increased levels of COL1 mRNA (2.3-fold, P<0.05) and proCOL1 protein (1.8-fold, P<0.05) in photoaged forearms to levels similar to that of young forearms. The results support the concept that reduced COL1 production in photoaged skin can be readily restored. This short-term regimen with ROL as a positive control can be used to evaluate the efficacy of potential COL1-enhancing topical agents.
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Enhanced dermal mechanical support rapidly stimulates fibroblasts and stable accumulation of collagen bundles in photoaged human skin F Wang, K Calderone, N Smith, T Do, S Kang, JJ Voorhees and G Fisher Department of Dermatology, University of Michigan, Ann Arbor, MI In photoaged human skin, type I collagen (COL) fibrils are fragmented, impairing dermal extracellular matrix (ECM) integrity, which collapses fibroblasts and reduces COL synthesis. Conversely, injecting cross-linked hyaluronic acid (CL-HA), a dermal filler, enhances dermal mechanical support, stimulating fibroblast synthesis of type I procollagen (PC1). We investigated the time course of these changes and whether stable accumulation of mature COL fibrils results. We injected CL-HA and vehicle (saline) into photoaged forearm skin of 31 human participants over 70 years old, and obtained biopsies. Starting at 1 week post-CL-HA injection, fibroblasts showed increased immunostaining of heat shock protein 47 (HSP47, 11-fold, p<.05, n¼15), a molecular chaperone required for PC1 synthesis. At this time point, gene expression of PC1, HSP47, and N-proteinase, an enzyme involved in processing of PC1 to COL, started increasing (76%, 31% and 69%, respectively; all p<.05, n¼14). As early as 4 weeks post-CL-HA injection, multiphoton microscopy showed elongated fibroblasts, indicating enhanced dermal mechanical support. Based on 3-D renders of images captured by second-harmonic generation microscopy, these fibroblasts were embedded within thick COL bundles densely packed around pools of injected CL-HA. HSP47 staining (n¼8) and gene expression of PC1, HSP47, and N-proteinase (each n¼9) remained elevated for as long as 6-9 months post-CL-HA injection (all p<.05). At 12 months post-injection, injected CL-HA was present in amounts similar to earlier time points and surrounded by thick collagen bundles. Thus, injected CL-HA rapidly (within 1-4 weeks) enhances mechanical support in the ECM microenvironment of fibroblasts, stimulating their elongation and synthesis of PC1, which is processed to thick, densely packed COL bundles. Whereas PC1 synthesis lasts as long as 6-9 months post-injection, both injected CL-HA and accumulated COL bundles persist for at least 12 months, likely causing prolonged clinical improvement.
Transcriptome analysis of psoriasis and wounded skin SX Chen, KA Chun, T Hata and RL Gallo University of California, San Diego, La Jolla, CA Significant similarities between psoriasis and wound healing have been demonstrated in vitro and in vivo, but no literature exists at the transcriptome level. Here, we use RNA-sequencing (RNA-seq) to compare psoriasis and wound healing to further elucidate the pathogenesis of psoriasis. Two psoriasis subjects received one 2 mm punch biopsy of a psoriatic plaque and two control subjects received one 2 mm punch biopsy of normal skin. Three days later, each control subject received a 2 mm punch biopsy over the initial biopsy site. RNA-seq of postwounded skin showed a transcriptome profile distinct from pre-wounded skin that more closely matched psoriasis expression in both heat map and hierarchical cluster analysis. To confirm this quantitatively, differentially expressed genes (DEGs) with >4 fold change and p<0.05 were identified. 52% of 810 psoriasis genes were implicated in healthy wounding. However, 1616 genes were identified in post-wounded skin. Gene ontology analysis for normal wounded skin and psoriatic lesional skin demonstrated greatest enrichment in keratinization. However, normal wounded skin showed unique enrichment for cell activation, leukocyte activation and adhesion, and inflammatory response. In contrast, psoriasis lesional skin was uniquely enriched in epithelial cell differentiation, and epidermis and ectoderm development. These findings suggest that psoriasis and normal wounding share similarities at the transcriptome level, but psoriasis may lack genes required for normal wound healing. Further investigations with larger sample sizes are necessary to confirm these preliminary findings.
www.jidonline.org S47
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Topical treatments improved periorbital aging J Namkoong, B Cook, D Kern and HE Knaggs Global R&D, Nu Skin Enterprises, Inc., Provo, UT Facial aging is manifested by different symptoms. One area people pay more attention to and use additional cosmetic products to keep healthier is the eye area e the periorbital region. There are multiple reasons why the periorbital region manifests an unhealthy and aged look. The skin in the eye area is thinner than other parts of face, and repetitive muscle movements and UV exposures lead to skin aging. Periorbital aging includes eyelid drooping, dark circles, under eye bags, or fine lines/wrinkles. Topical and mechanical treatments to improve periorbital aging is challenging due to the proximity to eyes. Hypersensitivity to treatment options are common occurrence in the eyes. In order to improve the skin in the eye area, topical ingredients were evaluated for their potential to modulate different skin aging target genes, such as improving skin structures, hydration, anti-inflammation, antioxidant capacity and cell turnover, using normal human skin equivalent cultures. Some interesting finding includes modulation of Prostaglandin E2 (PGE2). Induction of cellular stress with 5mM or 10mM hydrogen peroxide increased PGE2 expression, which would increase inflammation. In addition, PGE2 increase has been suggested in the skin of elderly and inhibition of PGE2 resulted in collagen production in skin organ culture. Experimental ingredients were able to suppress that H2O2-induced PGE2 expression, similar to ascorbic acid. After evaluating ingredients using in vitro models, a specific formula was developed to target clinical signs of periorbital skin aging. Prior to running a clinical study, the formula was evaluated for safety, due to potential hypersensitivity. This eye formulation did not trigger any safety concerns during in vitro evaluations and non-ocular evaluations. The formula was further assessed for safety during the efficacy testing by both dermatological and ophthalmologic evaluations. This formula improved periorbital aging in 50% of subjects with sagging skin, eyelid firmness and puffiness after 12 weeks of application by dermatological evaluations.
Pilot trial to evaluate the effect of vitamin D on melanocyte biomarkers E Anderson1, M Rezaee2, I Bailey-Healy2, K Sarin2 and J Tang2,1 1 School of Medicine, Stanford University, Stanford, CA and 2 Dermatology, Stanford University, Stanford, CA The role of vitamin D for cancer prevention is an expanding area of interest. In clinical and epidemiologic studies, vitamin D has been shown to predict incidence and severity of melanoma skin cancer. Although the role of vitamin D in melanocyte differentiation and melanoma carcinogenesis has been described, a clear mechanism is yet to be established. Previous studies have suggested that vitamin D supplementation and increased serum vitamin D levels are associated with decreased melanoma incidence. We sought to determine the effect of oral vitamin D supplementation on gene expression within melanocytic nevi of women previously diagnosed with non-melanoma skin cancer. A total of 24 healthy women were included in this randomized placebo-controlled study. Half of the cohort received 4,000 IU oral vitamin D3 daily for 8 weeks. Skin checks were performed at baseline and 8 week follow-up, and nevi were biopsied at both time points. RNA was collected from these nevi for gene expression profiling to identify activity in signaling pathways associated with vitamin D treatment. Women who received vitamin D supplementation experienced a significant increase in vitamin D levels at follow-up (p ¼ 0.0001) while women who received placebo did not (p ¼ 0.50). Gene expression analysis of melanocytic nevi revealed 1048 genes that were significantly differentially expressed between vitamin D-treated and placebo-controlled women. Ingenuity Pathway Analysis indicated that the most significantly differentiated molecular signaling pathways were related to immune system signaling, including primary immunodeficiency signaling (p ¼ 3.53 x 10-6), T cell receptor signaling (p ¼ 4.31 x 10-5), iCOS-iCOSL signaling in T helper cells (p ¼ 1.24 x 10-4), calcium-induced T lymphocyte apoptosis (p ¼ 7.52 x 10-4), and crosstalk between dendritic cells and natural killer cells (p ¼ 1.68 x 10-3). This finding suggests that increased immune surveillance could underlie the previously described association between vitamin D supplementation and decreased melanoma incidence.
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Growth of the cerebriform connective tissue nevus in individuals with Proteus syndrome N Nathan1, R Patel2, M Crenshaw2, M Lindhurst2, C Olsen3, L Biesecker2, K Keppler-Noreuil2 and T Darling1 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Medical Genomics and Metabolic Genetics Branch, NHGRI, Bethesda, MD and 3 Preventative Medicine and Biometrics, Uniformed Services University of the Health Sciences, Bethesda, MD The plantar cerebriform connective tissue nevus (CCTN) is the most common and problematic cutaneous manifestation of Proteus syndrome, an overgrowth syndrome caused by mosaicism for an activating mutation in AKT1. In order to gain insights into CCTN pathogenesis and natural history, we measured the size of plantar CCTNs using serial images from patients evaluated at the National Institutes of Health Clinical Center. The area of CCTN relative to total plantar area, excluding toes, were measured on 152 images from 22 patients (median age 6.9 years). The relative area of CCTN increased with age in children (n¼17, p<.0001) but not adults. Confluent small papules and nodules extending beyond the boundaries of CCTNs were gradually replaced by fully developed CCTN over time. The center of CCTN overlap from 14 patients appeared to be near the ball of the foot. This unexpected non-random distribution in a mosaic disorder may suggest that growth is augmented by minor trauma from walking. In 8 patients who underwent plantar CCTN biopsy and AKT1 mutational analysis, a positive relationship between CCTN growth rate and mutant allele frequency was observed (Spearman’s rho¼.62, p¼.10). Overall, these observations suggest that the progressive growth of CCTNs is impacted by multiple factors, including age, minor trauma, and the regional density of cells bearing AKT1 mutations.
Once weekly topical all-trans retinol restores type I collagen synthesis in photoaged forearm skin within 4 weeks: A protocol for evaluating anti-aging topical agents P Wang, M Sun, Y Xu, Y Xu, JJ Voorhees, G Fisher and Y Li Dermatology, University of Michigan, Ann Arbor, USA Minor Outlying Islands Type I collagen (COL1) is the major structural protein of human skin. COL1 content declines during aging, leading to skin thinning and fragility in the elderly. This collagen deficiency results in part from reduced COL1 synthesis manifested as reduced COL1 mRNA and type I procollagen (proCOL1) protein levels in aged skin. Daily use of topical retinoids, including all-trans retinol (ROL), has been shown to restore COL1 synthesis in the skin of the elderly. Typically, these studies have involved several months of daily treatment. How fast and to what extent topical ROL can restore COL1 synthesis in photoaged skin to that of young skin has not been well demonstrated. Herein, we investigated a short-term ROL treatment regimen. A oneday occlusion of ROL (0.4%) or vehicle was applied on photoaged forearms of elderly (>65 years old) subjects once a week for 4 weeks. Vehicle was also applied on forearms of young (23-33 years) subjects in the same manner. Skin samples were obtained one week post the last treatment and analyzed for COL1 levels. ROL treatment increased levels of COL1 mRNA (2.3-fold, P<0.05) and proCOL1 protein (1.8-fold, P<0.05) in photoaged forearms to levels similar to that of young forearms. The results support the concept that reduced COL1 production in photoaged skin can be readily restored. This short-term regimen with ROL as a positive control can be used to evaluate the efficacy of potential COL1-enhancing topical agents.
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Enhanced dermal mechanical support rapidly stimulates fibroblasts and stable accumulation of collagen bundles in photoaged human skin F Wang, K Calderone, N Smith, T Do, S Kang, JJ Voorhees and G Fisher Department of Dermatology, University of Michigan, Ann Arbor, MI In photoaged human skin, type I collagen (COL) fibrils are fragmented, impairing dermal extracellular matrix (ECM) integrity, which collapses fibroblasts and reduces COL synthesis. Conversely, injecting cross-linked hyaluronic acid (CL-HA), a dermal filler, enhances dermal mechanical support, stimulating fibroblast synthesis of type I procollagen (PC1). We investigated the time course of these changes and whether stable accumulation of mature COL fibrils results. We injected CL-HA and vehicle (saline) into photoaged forearm skin of 31 human participants over 70 years old, and obtained biopsies. Starting at 1 week post-CL-HA injection, fibroblasts showed increased immunostaining of heat shock protein 47 (HSP47, 11-fold, p<.05, n¼15), a molecular chaperone required for PC1 synthesis. At this time point, gene expression of PC1, HSP47, and N-proteinase, an enzyme involved in processing of PC1 to COL, started increasing (76%, 31% and 69%, respectively; all p<.05, n¼14). As early as 4 weeks post-CL-HA injection, multiphoton microscopy showed elongated fibroblasts, indicating enhanced dermal mechanical support. Based on 3-D renders of images captured by second-harmonic generation microscopy, these fibroblasts were embedded within thick COL bundles densely packed around pools of injected CL-HA. HSP47 staining (n¼8) and gene expression of PC1, HSP47, and N-proteinase (each n¼9) remained elevated for as long as 6-9 months post-CL-HA injection (all p<.05). At 12 months post-injection, injected CL-HA was present in amounts similar to earlier time points and surrounded by thick collagen bundles. Thus, injected CL-HA rapidly (within 1-4 weeks) enhances mechanical support in the ECM microenvironment of fibroblasts, stimulating their elongation and synthesis of PC1, which is processed to thick, densely packed COL bundles. Whereas PC1 synthesis lasts as long as 6-9 months post-injection, both injected CL-HA and accumulated COL bundles persist for at least 12 months, likely causing prolonged clinical improvement.
Transcriptome analysis of psoriasis and wounded skin SX Chen, KA Chun, T Hata and RL Gallo University of California, San Diego, La Jolla, CA Significant similarities between psoriasis and wound healing have been demonstrated in vitro and in vivo, but no literature exists at the transcriptome level. Here, we use RNA-sequencing (RNA-seq) to compare psoriasis and wound healing to further elucidate the pathogenesis of psoriasis. Two psoriasis subjects received one 2 mm punch biopsy of a psoriatic plaque and two control subjects received one 2 mm punch biopsy of normal skin. Three days later, each control subject received a 2 mm punch biopsy over the initial biopsy site. RNA-seq of postwounded skin showed a transcriptome profile distinct from pre-wounded skin that more closely matched psoriasis expression in both heat map and hierarchical cluster analysis. To confirm this quantitatively, differentially expressed genes (DEGs) with >4 fold change and p<0.05 were identified. 52% of 810 psoriasis genes were implicated in healthy wounding. However, 1616 genes were identified in post-wounded skin. Gene ontology analysis for normal wounded skin and psoriatic lesional skin demonstrated greatest enrichment in keratinization. However, normal wounded skin showed unique enrichment for cell activation, leukocyte activation and adhesion, and inflammatory response. In contrast, psoriasis lesional skin was uniquely enriched in epithelial cell differentiation, and epidermis and ectoderm development. These findings suggest that psoriasis and normal wounding share similarities at the transcriptome level, but psoriasis may lack genes required for normal wound healing. Further investigations with larger sample sizes are necessary to confirm these preliminary findings.
www.jidonline.org S47