- Email: [email protected]
322 Decline of the miRNA maturation machinery during skin aging
Epidermal Structure & Barrier Function | ABSTRACTS 321
322
Improvement of barrier impairment by topical application of a dual plasmin and urokinase inhibitor AV Rawlings1, P Wikstroem2, R Campiche2, E Jackson2, D Imfeld2 and R Voegeli2 1 AVR Consulting Ltd., Northwich, United Kingdom and 2 DSM Nutritional Products Ltd., Kaiseraugst, Switzerland We recently showed that the activities of the serine proteases of the plasminogen system (plasmin and urokinase) are elevated in facial stratum corneum (SC) compared to forearm SC and correlated with TEWL. We also demonstrated that plasmin activity is increased on sunexposed SC. Moreover, elevated plasmin activity was associated with reduced corneocyte maturation. The protease/protease-inhibitor balance of the plasminogen system may be key for the SC maturation processes and optimal barrier function. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application of barrier-impaired skin. Conventional synthetic, characterization and modelling methods were used in the development of the dual competitive inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide (BSFA) with Ki values 46 and 25 nM for plasmin and urokinase, respectively. The catalytic domain of both proteases is highly conserved and in both models BSFA showed a similar binding mode in silico. In a study on normal human keratinocytes stimulated with inflammatory cytokines (IL-1b, TNFa) the addition of BSFA (100mM) led to a down-regulation of both plasmin (40%) and urokinase (97%) activities. Additionally, an increased gene expression (4.5x) of transglutaminase 1 was induced by BSFA in the cytokine-stimulated keratinocytes. Finally, in a four week, vehicle-controlled study on 44 female Caucasian subjects the effect of a topically applied BSFA hydrogel formulation (10 ppm) was evaluated for basal function and recovery of the barrier of facial skin that was previously challenged by tape stripping. Compared to the vehicle treated group the BSFA treated group showed significantly improved SC barrier integrity, chemo-sensation to a lactic acid challenge and facial skin condition. Our data reveal that the topical application of an inhibitor of the plasminogen system is a novel approach for the treatment of barrier impaired skin and premature desquamation.
Decline of the miRNA maturation machinery during skin aging L Mur, A Lebleu, C Gondran, Y Ferreira, G Oberto, E Oger, L Bergeron, J Botto, K Cucumel and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Gaining a better understanding of the different processes and their regulations occurring during skin aging is fundamental to improve solutions to fight the signs of skin aging. The emergence of miRNA studies and their roles in post-transcriptional inhibition of gene expression highlights the importance of these small RNAs in the skin aging process. miRNA maturation is divided into two major steps: the primary miRNA is processed by Drosha to generate a pre-miRNA; and after exportation in the cytoplasm, Dicer allows the final maturation of miRNA. On senescent lung fibroblasts, it is proved that the expression of these two key markers decreases, involving the dysregulation of 20% of miRNAs. Among them, miRNA19b expression was shown to decrease with aging except for the centenarians, who keep a high level of this miRNA, suggesting its association with longevity. Firstly, we evaluated the expression of Drosha and Dicer by qPCR in in vitro skin fibroblasts that were aged by replicative senescence. We observed the decrease in the expression of these two key markers, as well as the decrease in miR-19b expression in senescent cells. In addition, we observed an over-expression of cyclin D1, one of miR-19b targets, in senescent cells, showing the effect of this miRdysregulation on the cell cycle. Secondly, in vitro fibroblasts were transfected with Dicer-siRNA. The partial inhibition of Dicer expression induced the increase in SA b-galactosidase activity and decreased in miR-19b expression. Finally, we tested an innovative baobab seed extract rich in small RNAs on these two senescent models. The treatment with this extract counteracted the negative effects on the dysregulation of miRNA maturation machinery observed on senescent fibroblasts. To complete these in vitro results, a clinical test was performed on wrinkle appearance and skin moisturization. Our present results suggest that the maintenance of miRNA maturation machinery is essential to minimize senescence and thereby to limit the appearance of skin aging.
323
324
A parathyroid hormone family member TIP39 increases intracellular calcium via the IP3 pathway E Sato1, T Takahashi1, TB Usdin2 and RL Gallo1 1 Dermatology, University of California, San Diego, San Diego, CA and 2 National Institute of Mental Health, Bethesda, MD Parathyroid hormone (PTH) acts with Vitamin D to control serum calcium by increasing bone resorption in bone, intestine and kidney. Furthermore, some evidence exists that PTH directly increases intercellular calcium ([Ca2+]i) via both cAMP and inositol 1,4,5-triphosphate (IP3) pathway in various cells. We previously reported that keratinocytes express a novel member of the PTH family known as TIP39, and that addition of recombinant TIP39 to normal human keratinocytes (NHEK) induced an increase in [Ca2+]i and triggered aspects of terminal differentiation including decreased keratin-14 and increased involucrin expression. We now show that TIP39 increases [Ca2+]i via its receptor PTH2R, phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Under low concentrations of extracellular calcium ([Ca2+]e), PTH2R knockdown decreased TIP39 effects on [Ca2+]i of passage 6 (p6) NHEK from 7.0% to 1.2% (p<0.005). In the case of p1 NHEK, 10mM TIP39 increased [Ca2+]i 17.8% compared with the control group. The pretreatment of inhibitors of IP3Rs, PLC and calmodulin reduced the effects of TIP39 from 17.8% to 5.7%, 6.0% and 7.1% respectively. Thapsigargin, which is an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), did not inhibit TIP39 responses compared with those inhibitors (12.2%). These data suggest TIP39 enhances keratinocyte Ca2+ uptake and suggests the novel hypothesis that this molecule may also play a role in genetic skin disorders demonstrating an abnormality of calcium transport such as Darier disease or Hailey-Hailey disease.
Bioinformatic modeling of the interaction network of genes implicated in skin senescence C Serre, L Bergeron, C Capallere, C Plaza, C Meyrignac, N Esselin, V Busuttil, J Botto and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Cellular senescence is an irreversible state of cell cycle arrest that is induced during cellular aging. In the skin, senescence is associated with phenotypic changes in cutaneous structure and cells, lowered resistance to oxidative stress and DNA damage, decreased epidermal cell renewal, decreased synthesis of extracellular matrix protein and the appearance of markers of aging (senescence-associated beta-galactosidase, etc.). Many genes have been reported to be involved in the regulation of senescence: tumor suppressor genes (e.g., p53), senescence genes (e.g., p16, p21) and senescence suppressor genes (e.g., telomerase), oncogenes (e.g., MYC), stemness genes (e.g., SOX2), genes related with epigenetics (e.g., SIRT1), inflammation-related genes, genes implicated in DNA damage repair, cytoskeletal remodeling (e.g., TGF, WNT), and also various transcription factors. All these genes participate in a network of interactions regulating senescence, ending up with the highly regulated p53ep21 and p16epRB pathways. In this study we developed a bioinformatic network model of the relationships between the main genes involved in skin senescence. This network allowed the categorization of subsets of genes and to predict potential modulating microRNAs. In parallel, we developed several in vitro models to study senescence, based on the silencing of specific genes allowing to obtain skin cells in culture with a senescent phenotype. Characterization of the cellular phenotype by flow cytometry, expression of proteic markers and qPCR was undertaken in order to point out specific features according to the gene silenced. This complementary in silico and in vitro testing approach will facilitate the study of the modulating potential of biofunctional ingredients or chemicals on the cellular senescence in vitro.
325
326
Biodegradable microneedle patch to increase the penetration of topical steroid in the skin J Shin, H Kang, J Lee, H Chu, H Lee and K Lee Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Background: Although topical steroid is the first choice of drug in prurigo nodularis, most patients do not sufficiently respond to the strong steroids possibly due to the low penetration rate of drugs in thickened skin. Objective: This study was performed to investigate if the applying of biodegradable microneedle patch after the use of topical steroid increases the penetration of topical steroids and treatment efficacy in prurigo nodularis patients. Methods: The penetration rate of topical steroid was measured by Franz diffusion cell system using 3D skin. Patients who have prurigo nodularis in both side of arms or legs were included. Right arm was treated with biodegradable microneedle patch after topical steroid and left arm was treated with topical steroid only. The height and area of each skin lesion was measured by independent dermatologists. Patients evaluated treatment response and visual analog scale (VAS). Dermatologist also evaluated patients’ skin lesions using investigator’s global assessment score (IGA). Result: The penetration of topical steroid was significantly higher in biodegradable microneedle applied 3D skin equivalent than non-applied 3D skin equivalent. Area and height of prurigo nodularis decreased after 4 weeks of treatment in both biodegradable microneedle applied and non-applied side. However, biodegradable microneedle patch applied side showed more decrease in area and height of prurigo nodularis compared to non-applied side (P < 0.05). Visual analog scale (VAS) was also significantly lower in biodegradable microneedle applied side (p < 0.05). Conclusion: Our results demonstrated that applying of biodegradable microneedle patch after the use of topical steroid can enhance the penetration of topical steroids and increase the treatment efficacy. This novel patch can be used efficiently in clinical field given its patient safety and effectiveness in prurigo nodularis treatment.
Two methods of keratinocyte extraction: Do they involve two different types of keratinocyte behavior? G Bressier, M Bonnans, N Garcia, C Plaza, C Capallere, J Botto and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Routine methods set to isolate and cultivate human primary keratinocytes were first developed in the 1970s by Rheinwald and Green. As many steps may differ between isolation methods, it is of major concern to ensure that isolated keratinocyte behavior is the same regardless of the isolation process. The objective of this study was to compare two different methods of keratinocyte extraction used in our laboratories. Extracted cells were studied regarding growth, development and reactivity patterns. Cells were isolated from human donors, following the two protocols, and cultivated in monolayer. In the first step, we focused on the observation of keratinocyte morphology as well as different markers characteristic of their life cycle and differentiation. To complete this study and observe the impact of aging on their evolution, two subcultures of keratinocytes were performed. Following our observations in term of morphology and development, we focused in the second step on keratinocyte reactivity. For this study cells were treated with and without chemicals that are well known for their impact on cell activity. Results obtained from three different donors showed that, irrespective of the subculture, keratinocytes had grown in clusters for one isolation process, or as individual cells with the other one. However, regarding the evaluation of cell cohesion and differentiation using b1-integrin, KLK7, involucrin, and Pankeratin immunodetections, it appeared that they presented nearly the same pattern of expression. In the second step, keratinocytes showed the same reactivity when stimulated with positive controls. However, we highlighted that the level of differentiation was slightly higher for one pool of extracted keratinocytes, suggesting that these cells should not be used with numerous subcultures for late differentiation observations. This study demonstrated that whatever the isolation process used, keratinocytes behave the same way regarding their proliferation and differentiation.
www.jidonline.org S57
322
Improvement of barrier impairment by topical application of a dual plasmin and urokinase inhibitor AV Rawlings1, P Wikstroem2, R Campiche2, E Jackson2, D Imfeld2 and R Voegeli2 1 AVR Consulting Ltd., Northwich, United Kingdom and 2 DSM Nutritional Products Ltd., Kaiseraugst, Switzerland We recently showed that the activities of the serine proteases of the plasminogen system (plasmin and urokinase) are elevated in facial stratum corneum (SC) compared to forearm SC and correlated with TEWL. We also demonstrated that plasmin activity is increased on sunexposed SC. Moreover, elevated plasmin activity was associated with reduced corneocyte maturation. The protease/protease-inhibitor balance of the plasminogen system may be key for the SC maturation processes and optimal barrier function. Thus, our aim was to develop a specific dual plasmin and urokinase inhibitor for topical application of barrier-impaired skin. Conventional synthetic, characterization and modelling methods were used in the development of the dual competitive inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide (BSFA) with Ki values 46 and 25 nM for plasmin and urokinase, respectively. The catalytic domain of both proteases is highly conserved and in both models BSFA showed a similar binding mode in silico. In a study on normal human keratinocytes stimulated with inflammatory cytokines (IL-1b, TNFa) the addition of BSFA (100mM) led to a down-regulation of both plasmin (40%) and urokinase (97%) activities. Additionally, an increased gene expression (4.5x) of transglutaminase 1 was induced by BSFA in the cytokine-stimulated keratinocytes. Finally, in a four week, vehicle-controlled study on 44 female Caucasian subjects the effect of a topically applied BSFA hydrogel formulation (10 ppm) was evaluated for basal function and recovery of the barrier of facial skin that was previously challenged by tape stripping. Compared to the vehicle treated group the BSFA treated group showed significantly improved SC barrier integrity, chemo-sensation to a lactic acid challenge and facial skin condition. Our data reveal that the topical application of an inhibitor of the plasminogen system is a novel approach for the treatment of barrier impaired skin and premature desquamation.
Decline of the miRNA maturation machinery during skin aging L Mur, A Lebleu, C Gondran, Y Ferreira, G Oberto, E Oger, L Bergeron, J Botto, K Cucumel and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Gaining a better understanding of the different processes and their regulations occurring during skin aging is fundamental to improve solutions to fight the signs of skin aging. The emergence of miRNA studies and their roles in post-transcriptional inhibition of gene expression highlights the importance of these small RNAs in the skin aging process. miRNA maturation is divided into two major steps: the primary miRNA is processed by Drosha to generate a pre-miRNA; and after exportation in the cytoplasm, Dicer allows the final maturation of miRNA. On senescent lung fibroblasts, it is proved that the expression of these two key markers decreases, involving the dysregulation of 20% of miRNAs. Among them, miRNA19b expression was shown to decrease with aging except for the centenarians, who keep a high level of this miRNA, suggesting its association with longevity. Firstly, we evaluated the expression of Drosha and Dicer by qPCR in in vitro skin fibroblasts that were aged by replicative senescence. We observed the decrease in the expression of these two key markers, as well as the decrease in miR-19b expression in senescent cells. In addition, we observed an over-expression of cyclin D1, one of miR-19b targets, in senescent cells, showing the effect of this miRdysregulation on the cell cycle. Secondly, in vitro fibroblasts were transfected with Dicer-siRNA. The partial inhibition of Dicer expression induced the increase in SA b-galactosidase activity and decreased in miR-19b expression. Finally, we tested an innovative baobab seed extract rich in small RNAs on these two senescent models. The treatment with this extract counteracted the negative effects on the dysregulation of miRNA maturation machinery observed on senescent fibroblasts. To complete these in vitro results, a clinical test was performed on wrinkle appearance and skin moisturization. Our present results suggest that the maintenance of miRNA maturation machinery is essential to minimize senescence and thereby to limit the appearance of skin aging.
323
324
A parathyroid hormone family member TIP39 increases intracellular calcium via the IP3 pathway E Sato1, T Takahashi1, TB Usdin2 and RL Gallo1 1 Dermatology, University of California, San Diego, San Diego, CA and 2 National Institute of Mental Health, Bethesda, MD Parathyroid hormone (PTH) acts with Vitamin D to control serum calcium by increasing bone resorption in bone, intestine and kidney. Furthermore, some evidence exists that PTH directly increases intercellular calcium ([Ca2+]i) via both cAMP and inositol 1,4,5-triphosphate (IP3) pathway in various cells. We previously reported that keratinocytes express a novel member of the PTH family known as TIP39, and that addition of recombinant TIP39 to normal human keratinocytes (NHEK) induced an increase in [Ca2+]i and triggered aspects of terminal differentiation including decreased keratin-14 and increased involucrin expression. We now show that TIP39 increases [Ca2+]i via its receptor PTH2R, phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Under low concentrations of extracellular calcium ([Ca2+]e), PTH2R knockdown decreased TIP39 effects on [Ca2+]i of passage 6 (p6) NHEK from 7.0% to 1.2% (p<0.005). In the case of p1 NHEK, 10mM TIP39 increased [Ca2+]i 17.8% compared with the control group. The pretreatment of inhibitors of IP3Rs, PLC and calmodulin reduced the effects of TIP39 from 17.8% to 5.7%, 6.0% and 7.1% respectively. Thapsigargin, which is an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), did not inhibit TIP39 responses compared with those inhibitors (12.2%). These data suggest TIP39 enhances keratinocyte Ca2+ uptake and suggests the novel hypothesis that this molecule may also play a role in genetic skin disorders demonstrating an abnormality of calcium transport such as Darier disease or Hailey-Hailey disease.
Bioinformatic modeling of the interaction network of genes implicated in skin senescence C Serre, L Bergeron, C Capallere, C Plaza, C Meyrignac, N Esselin, V Busuttil, J Botto and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Cellular senescence is an irreversible state of cell cycle arrest that is induced during cellular aging. In the skin, senescence is associated with phenotypic changes in cutaneous structure and cells, lowered resistance to oxidative stress and DNA damage, decreased epidermal cell renewal, decreased synthesis of extracellular matrix protein and the appearance of markers of aging (senescence-associated beta-galactosidase, etc.). Many genes have been reported to be involved in the regulation of senescence: tumor suppressor genes (e.g., p53), senescence genes (e.g., p16, p21) and senescence suppressor genes (e.g., telomerase), oncogenes (e.g., MYC), stemness genes (e.g., SOX2), genes related with epigenetics (e.g., SIRT1), inflammation-related genes, genes implicated in DNA damage repair, cytoskeletal remodeling (e.g., TGF, WNT), and also various transcription factors. All these genes participate in a network of interactions regulating senescence, ending up with the highly regulated p53ep21 and p16epRB pathways. In this study we developed a bioinformatic network model of the relationships between the main genes involved in skin senescence. This network allowed the categorization of subsets of genes and to predict potential modulating microRNAs. In parallel, we developed several in vitro models to study senescence, based on the silencing of specific genes allowing to obtain skin cells in culture with a senescent phenotype. Characterization of the cellular phenotype by flow cytometry, expression of proteic markers and qPCR was undertaken in order to point out specific features according to the gene silenced. This complementary in silico and in vitro testing approach will facilitate the study of the modulating potential of biofunctional ingredients or chemicals on the cellular senescence in vitro.
325
326
Biodegradable microneedle patch to increase the penetration of topical steroid in the skin J Shin, H Kang, J Lee, H Chu, H Lee and K Lee Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea (the Republic of) Background: Although topical steroid is the first choice of drug in prurigo nodularis, most patients do not sufficiently respond to the strong steroids possibly due to the low penetration rate of drugs in thickened skin. Objective: This study was performed to investigate if the applying of biodegradable microneedle patch after the use of topical steroid increases the penetration of topical steroids and treatment efficacy in prurigo nodularis patients. Methods: The penetration rate of topical steroid was measured by Franz diffusion cell system using 3D skin. Patients who have prurigo nodularis in both side of arms or legs were included. Right arm was treated with biodegradable microneedle patch after topical steroid and left arm was treated with topical steroid only. The height and area of each skin lesion was measured by independent dermatologists. Patients evaluated treatment response and visual analog scale (VAS). Dermatologist also evaluated patients’ skin lesions using investigator’s global assessment score (IGA). Result: The penetration of topical steroid was significantly higher in biodegradable microneedle applied 3D skin equivalent than non-applied 3D skin equivalent. Area and height of prurigo nodularis decreased after 4 weeks of treatment in both biodegradable microneedle applied and non-applied side. However, biodegradable microneedle patch applied side showed more decrease in area and height of prurigo nodularis compared to non-applied side (P < 0.05). Visual analog scale (VAS) was also significantly lower in biodegradable microneedle applied side (p < 0.05). Conclusion: Our results demonstrated that applying of biodegradable microneedle patch after the use of topical steroid can enhance the penetration of topical steroids and increase the treatment efficacy. This novel patch can be used efficiently in clinical field given its patient safety and effectiveness in prurigo nodularis treatment.
Two methods of keratinocyte extraction: Do they involve two different types of keratinocyte behavior? G Bressier, M Bonnans, N Garcia, C Plaza, C Capallere, J Botto and N Domloge Ashland Specialty Ingredients, Sophia Antipolis, France Routine methods set to isolate and cultivate human primary keratinocytes were first developed in the 1970s by Rheinwald and Green. As many steps may differ between isolation methods, it is of major concern to ensure that isolated keratinocyte behavior is the same regardless of the isolation process. The objective of this study was to compare two different methods of keratinocyte extraction used in our laboratories. Extracted cells were studied regarding growth, development and reactivity patterns. Cells were isolated from human donors, following the two protocols, and cultivated in monolayer. In the first step, we focused on the observation of keratinocyte morphology as well as different markers characteristic of their life cycle and differentiation. To complete this study and observe the impact of aging on their evolution, two subcultures of keratinocytes were performed. Following our observations in term of morphology and development, we focused in the second step on keratinocyte reactivity. For this study cells were treated with and without chemicals that are well known for their impact on cell activity. Results obtained from three different donors showed that, irrespective of the subculture, keratinocytes had grown in clusters for one isolation process, or as individual cells with the other one. However, regarding the evaluation of cell cohesion and differentiation using b1-integrin, KLK7, involucrin, and Pankeratin immunodetections, it appeared that they presented nearly the same pattern of expression. In the second step, keratinocytes showed the same reactivity when stimulated with positive controls. However, we highlighted that the level of differentiation was slightly higher for one pool of extracted keratinocytes, suggesting that these cells should not be used with numerous subcultures for late differentiation observations. This study demonstrated that whatever the isolation process used, keratinocytes behave the same way regarding their proliferation and differentiation.
www.jidonline.org S57