OBJECTIVE:Collagens are widely studied proteins given their implications in the skin extracellular matrix and pathological conditions such as fibrosis. Type V collagen is a member of the fibrillar collagens, and three different polypeptide chains, α1, α2 and α3 form isoforms through associations. The third chain, α3, of type V collagen was initially identified in the placenta, but after decades, it remains poorly characterized. The aim of this study is to investigate the expression and localization of the α3 chain of type V collagen in the skin and its evolution during skin aging. The identification of α3(V) collagen as a target of retinoic acid and its interaction with matrix metalloproteinase-9 (MMP-9) was investigated. METHODS:The expression and localization of α3(V) collagen were assessed by immunodetection in ex vivo skin and in keratinocytes using a calcium-differentiated model. The variation in α3(V) collagen expression during aging was studied in ex vivo skin from donors of different ages. The modulation of α3(V) collagen by retinoic acid was investigated by qPCR in keratinocytes and by immunofluorescence in ex vivo skin biopsies. Silencing of α3(V) collagen was performed using siRNA. The expression of MMP-9 was investigated using qPCR. RESULTS:In our study, we showed that α3(V) collagen was abundantly produced by basal skin keratinocytes and in the outer root sheath of the hair. α3(V) collagen expression appears to decrease with age in the skin and in differentiated keratinocytes. We also identified that treatment of keratinocytes and ex vivo skin biopsies with retinoic acid induced α3(V) collagen expression. Finally, we showed that inhibition of α3(V) collagen in keratinocytes led to an increase in MMP-9 expression. CONCLUSION:This study provides a better understanding of α3(V) collagen expression and localization in the skin. These findings suggest that α3(V) collagen could contribute to the therapeutic and anti-aging value of retinoic acid on the skin and may open new prospects. Finally, our data shed more light on the functional relationship between α3(V) collagen and MMP-9, opening possibilities for anti-aging and eczema treatments.

Objective: Throughout our existence, the skin senses and analyses the mechanical forces imposed by the environment. In response to these environmental forces, skin can deform itself and achieve a biological response. The subsequent cutaneous plasticity emerges from mechanical properties arising from the collective action of the skin cells, particularly keratinocytes, that govern the tensile strength via cell-to-cell adhesions and via cell-matrix adhesion structures. In addition to serving as force-bearing entities, keratinocytes respond to forces by activating signalling pathways to control their own fate and function. To detect and adapt to mechanical signals, keratinocytes possess a panel of sensory receptors and junctional intercellular structures. Mechanically activated ion channel Piezo1 has been described as a force sensor and as being involved in pleasant touch perception. In this study, relationships between Piezo1 modulation and oxytocin synthesis were investigated.Methods: The expression of Piezo1 in the skin was studied and compared with the expression of TRPV1. Dooku1 antagonist and Jedi1 agonist were used to modulate Piezo1. The level of E-cadherin and oxytocin was monitored in ex vivo skin biopsies by immunodetection.Results: Taken together, our results illustrate the major role of mechanosensitive ion channel Piezo1 in skin barrier integrity, and in peripheral oxytocin synthesis in the skin.Conclusion: In conclusion, this study highlights the relationships between pleasant touch, soft touch and local oxytocin synthesis.

OBJECTIVE:Air pollution is today fully acknowledged to be a significant public health problem. Rapid urbanization exposed us to a variety of unhealthy ambient air pollutants at high concentrations. The emergence of airborne ultrafine particles has added an additional dimension to this already complex problem of air pollution. The skin has different functions, one of them being the protection against the deleterious effect of external agents. The aim of this study is to evaluate the impact of airborne ultrafine particles (UFP) pollution on skin aging and on keratinocyte differentiation. METHODS:Ex vivo human skin biopsies and cultured keratinocytes stem cells (KSC) were submitted to diesel exhaust-derived UFP. Reactive oxygen species (ROS) production was assessed with the MitoSOX™ probe. Keratinocyte stemness potential was evaluated by the immunodetection of keratin 15 (K15) and p63 (∆N isoforms). Effect of UFP on the epithelial niche maintenance was evaluated by immunodetection of Sox9. Reconstructed epidermis model was used to assess the impact of UFP on keratinocyte differentiation and aging. RESULTS:UFP exposure induced ROS production and disturbed K15, ∆Np63 and Sox9 expression in KSC or ex vivo skin. Finally, investigations on reconstructed epidermis revealed a phenotype marked by impaired keratinocyte differentiation. CONCLUSION:These results indicate that UFP pollution is a potent extrinsic factor of skin aging, affecting the keratinocyte stem cell potential and the skin renewal process.

In skin, mechanisms exist to sense, transduce, and transmit force including mechanosensitive ion channels and E-cadherin-based cell-cell adhesions. Keratinocytes express the mechanoreceptor piezo1 and mediate touch sensation by detecting and encoding tactile information to sensory neurons. Moreover, pleasant touch has been shown to trigger oxytocin release, a molecule linked to increased levels of social interaction, well-being and anti-stress effects. In this study, a 100% natural J. Grandiflorum extract, was developed with a new to the world technology, inspired from natural plant-microorganisms interaction. The extraction process uses the phytobiome of jasmine flowers to orchestrate phytocompounds biotransformation, without any addition of exogenous microorganisms. Analytical investigations highlighted the uniqueness of the chemical profile and the richness of bioactive phytocompounds identified such as specific plant peptides – called jasmintides, organics acids and phenolic compounds. The extract was designed to mediate piezo1 activation, to mimic pleasant touch and promote release of oxytocin. Expression of piezo1, E-cadherin, and oxytocin were monitored by immunohistochemistry and ELISA assay. A clinical study was designed to evaluate the well-being and well-aging. Inex vivo skin, piezo1 blockage caused epidermal sagging, structural damages, and showed a decrease of E-cadherin and oxytocin levels. J. Grandiflorum extract preserved the skin morphology, maintained E-cadherin and oxytocin levels. The clinical study revealed an improvement of the overall well-being, salivary oxytocin level and emotional state after application of the extract. Our research suggests that the application of J. Grandiflorum extract could mimic pleasant touch, thought the activation of piezo1 channel in the skin; and activate the oxytocinergic system, including its powerful anti-aging effect, and promoting the expression of pleasant emotions.

ObjectiveThe skin is a sensory organ, densely innervated with various types of sensory nerve endings, capable of discriminating touch, environmental sensations, proprioception, and physical affection. Neurons communication with skin cells confer to the tissue the ability to undergo adaptive modifications during response to environmental changes or wound healing after injury. Thought for a long time to be dedicated to the central nervous system, the glutamatergic neuromodulation is increasingly described in peripheral tissues. Glutamate receptors and transporters have been identified in the skin. There is a strong interest in understanding the communication between keratinocytes and neurons, as the close contacts with intra-epidermal nerve fibers is a favorable site for efficient communication. To date, various coculture models have been described. However, these models were based on non-human or immortalized cell line. Even the use of induced pluripotent stem cells (iPSCs) is posing limitations because of epigenetic variations during the reprogramming process. MethodsIn this study, we performed small molecule-driven direct conversion of human skin primary fibroblasts into induced neurons (iNeurons). ResultsThe resulting iNeurons were mature, showed pan-neuronal markers, and exhibited a glutamatergic subtype and C-type fibers characteristics. Autologous coculture of iNeurons with human primary keratinocytes, fibroblasts, and melanocytes was performed and remained healthy for many days, making possible to study the establishment of intercellular interactions. ConclusionHere, we report that iNeurons and primary skin cells established contacts, with neurite ensheathment by keratinocytes, and demonstrated that iNeurons cocultured with primary skin cells provide a reliable model to examine intercellular communication.

OBJECTIVE:The epidermis possesses the capacity to replace dying cells and to heal wounds, thanks to resident stem cells, which have self-renewal properties. In skin physiology, miRNAs have been shown to be involved in many processes, including skin and hair morphogenesis. Recently, differentiation of epidermal stem cells was shown to be promoted by the miR-203. The miR-203 is upregulated during epidermal differentiation and is of interest because of significant targets.METHODS:By utilizing a bioinformatic tool, we identified a target site for miR-203 in the survivin mRNA. Silencing miR-203 was managed with the use of antagomir; the silencing of survivin was performed with a siRNA. Survivin expression was determined by qPCR or immunofluorescence in cultured cells, and by immunohistochemistry in skin sections. Involucrin expression was used as marker of keratinocyte differentiation. A rice extract with previously demonstrated anti-aging properties was evaluated on miR-203 modulation.RESULTS:In this study, we identified a miR-203/survivin axis, important for epidermal homeostasis. We report that differentiation of keratinocyte is dependent on the level of miR-203 expression and that inhibition of miR-203 can increase the expression of survivin, an epidermal marker of stemness.CONCLUSION:In summary, our findings suggest that miR-203 target 3'UTR region of survivin mRNA and directly represses survivin expression in the epidermis. The rice extract was identified as modulator of miR-203 and pointed out as a promising microRNA-based strategy in treating skin changes occurring with aging.

Skin functions as a neuro-immuno-endocrine tissue with well-defined neuronal networks and functions. The endocannabinoid system has been proven to be an important, homeostatic regulator for homeostatic and inflammatory events. The system comprises endogenous or exogenous ligands and receptors (CB1 and CB2). In the present study, we evaluated the soothing properties of a Pogostemon cablin (patchouli) extract. Agonist AM1241 and antagonist AM630 were used for CB2 receptor activation/inhibition. Expression of CB2 receptor and β-endorphin was monitored by immunohistochemistry. Skin inflammation was induced with ultraviolet B (UVB) or lipopolysaccharide (LPS), and the following markers were used to highlight the anti-inflammatory properties of the extract: transient receptor potential vanilloid 1 (TRPV1), interleukin receptors 1 (IL1R1), and the interleukin 6 signal transducer (IL6ST). Our results demonstrated the implication of the CB2 receptor in the skin inflammation process. The expression of CB2 receptor and β-endorphin was increased 48 hours after application of the extract. Furthermore, patchouli extract application helped to reduce IL1R1, IL6ST, and TRPV1 expression, in skin exposed to UVB or LPS. In conclusion, the application of the patchouli extract helps maintain skin integrity and reduce skin discomfort via modulation of CB2 receptor stimulation and the subsequent β-endorphin release.

Opsin photoreceptors are responsible for the absorption of light, transduce information about daily lighting conditions and provide vision. Opsins are also involved in diverse non-visual functions such as the circadian clock. The non-visual opsin 3, also known as encephalopsin, is expressed in skin and its functions remain poorly defined. The expression of opsin 3 in human skin tissue was evaluated by immunohistochemistry, in keratinocytes and melanocytes by immunodetection. Opsin 3 and β-tubulin colocalization was performed during the keratinocyte mitotic phase. Opsin 3-siRNA transfection was used in primary keratinocytes and successful transfection was confirmed by immunodetection. These results show that opsin 3 was detected in the epidermis of human skin, both in keratinocytes and melanocytes. Opsin 3 colocalize with β-tubulin during the mitotic phase and participate in keratinocyte cell division. Opsin 3 protein expression was reduced after transfection with opsin 3-siRNA; inhibition of opsin 3-siRNA reduces keratinocyte cell proliferation and promotes the apparition of binucleated cells. This study highlights an unconventional function of opsin 3 in extra-ocular cells. Opsin 3 appears to be involved in the regulation of late cytokinesis in keratinocytes, and therefore in the maintenance of epidermal homeostasis.

The skin has its own cannabinoid system which is part of its neuro-endocrine system. This system participates in various functions such as proliferation, differentiation, inflammation as well as the control of pain and itch sensation. Whereas CB1 is predominantly expressed in the central nervous system and peripheral tissues, CB2 was found mainly in non-neuronal tissues. The effects on CB2 have been previously reported as a potential target for reducing the sensation of skin discomfort, without psycho-active effects. In particular, CB2 receptor stimulation leads to beta-endorphin release from keratinocytes, that exhibits anti-nociceptive effects. Furthermore, cannabinoids can interact with other receptors associated with itch sensation and sensitive skin such as TRPV1. The present study aims to evaluate the effect of an extract of Pogostemon cablin (patchouli) on CB2 expression on ex vivo skin biopsies. This extract is obtained by a supercritical CO2 extraction of patchouli leaves and contains volatile molecules, including phytocannabinoids, as well as flavonoids, phytosterols and fatty acids. The effects on IL1-R1 (IL1 receptor) and TRPV1 were also investigated on ex vivo skin biopsies exposed to UVB irradiation or bacterial lipopolysaccharide (LPS), after application of a formula containing the patchouli extract. The level of CB2 expression in skin biopsies was observed to have increased after application of the extract. In skin biopsies with an increased inflammatory level, induced either by UVB or LPS, the application of the extract was associated with a reduction of the level of IL1-R1 and TRPV1. The ECS is one of the system the skin uses to respond to diverse stressors that lead to unpleasant skin sensation. The increase in CB2 expression, and the reduction of the inflammatory level and of TRPV1 associated with sensitive skin sensation are in favor of a decrease in the perceivable signs of skin discomfort.

Skin repair is a multistep process involving both epidermal and dermal compartments. Among the sequential and overlapping events that take place during the skin repair process, the regeneration of the epidermal compartment represents a critical step for a complete and effective repair, described here as “perfect skin repair”. In the present study, we evaluated an extract of Rosa centifolia, prepared using a proprietary extraction progress, that is rich in small RNA and compounds of interest including. This extract was evaluated for its effects on markers associated with skin repair, on normal aged human keratinocytes. MARCKS like protein (MARCKSL1) is a regeneration-initiating protein, discovered in axolotl, an amphibian species known for its scar-free healing capacity. MARCKSL1 is also expressed in human skin. Among the different miRNAs implicated in skin repair, miR-132 has been described as critical to improve skin regeneration, playing a role in the transition between the inflammatory phase and epidermal restoration. In the model of induced cellular senescence, we observed a decrease in MARCKSL1 and miR-132 expressions and a slowdown of cell migration in a scratch test assay. When senescent keratinocytes were treated with the extract of Rosa centifolia, the marker of perfect skin repair MARCKSL1, as well as the level of miR-132, were observed to have increased. The ability of senescent keratinocytes to regenerate a scratched monolayer was also observed to have improved after application of the extract. Some dermal markers associated with skin repair such as collagen I, fibronectin and hyaluronic acid were increased in ex vivo skin biopsies that had been treated with the extract. In conclusion, the extract of Rosa centifolia was shown to act on markers of the early phase of skin repair, associated with epidermal restoration, as well as components of the extracellular matrix. Taken together, these effects are in favor of optimizing skin repair especially in aged or compromised skin.

Exposure to ultrafine particles (UFP; ≤0.1 μm) occurs every day mainly from vehicle emissions Exposure of the skin to air pollutants (such as UFP) has been associated with skin aging and facial age spots, and the aggravation of some skin diseases. In the present study, we evaluated the protective effect against UFP-induced pollution exposure of a fraction of Macrocystis pyrifera (Giant Kelp extract). UFP were tested on in vitro cell culture (Rapidly Adherent (RA) keratinocytes), ex vivo skin biopsies and reconstructed human epidermis model. Reactive Oxygen Species (ROS), barrier function and hydration were monitored after UFP exposure. Keratinocyte stemness potential and epithelial niche maintenance were evaluated by keratin 15, p63 and SOX9. Skin hydration was also monitored in vivo by investigating hydration and corneodesmosin. Our results showed that UFP exposure increased ROS production in keratinocytes and caused a decrease in the expression of keratin 15, p63 and SOX9 in RA-keratinocytes. Moreover, UFP exposure affected loricrin expression in reconstructed human epidermis. We showed that Macrocystis pyrifera extract application was associated with a reduction in ROS production in keratinocytes, a preservation of stem cell and niche related markers, as well as an improvement of skin regenerative potential. After 7 days of applying the extract-containing cream, skin hydration was observed to have increased significantly compared to the placebo. Moreover, a peripheric staining of the corneodesmosin was observed in corneocytes from volunteers with enhanced skin hydration. This study pointed out UFP effect on epidermal renewal capacity. Despite the limited amount of data reporting UFP effects on skin, it has become more obvious that prolonged air pollution exposure is mainly responsible for the appearance of visible signs of premature skin aging.

The significance of Coenzyme Q10 (CoQ10) as an anti-oxidant barrier of the skin, as well as a key component in anti-aging strategies for skin care products, has been firmly established. Biosynthesis of CoQ10 in the mitochondria is well known, but there is only limited information on the non-mitochondrial synthesis of CoQ10 in the skin. Recent findings in zebrafish identified that a tumor suppressor, Ubiad1, is also a key enzyme in the non-mitochondrial synthesis of CoQ10. The purpose of this study was to investigate expression of Ubiad1 in human skin, and its implication in the skin’s cutaneous response to oxidative stress. We observed Ubiad1 localization in the epidermis, particularly a subcellular localization in the Golgi apparatus. Ubiad1 modulation by a pentapeptide was associated with an observed reduction in ROS/RNS stresses (−44%/−19% respectively), lipid peroxidation (−25%) and preservation of membrane fluidity under stress conditions. Electron microscopy of keratinocytes revealed a significant degree of stimulation of the Golgi complex, as well as significantly improved mitochondrial morphology. Given the importance of CoQ10 in mitigating the visible signs of skin aging, our findings identify Ubiad1 as an essential component of the defensive barriers of the epidermis.

Ultraviolet rays, pollutants and the resulting oxidative stress all contribute significantly to the appearance of skin aging. Human skin cells' ability to repair oxidative damage steadily decreases over the years, leading to reactive oxygen species (ROS) accumulation. Over time, the presence of lipophilic antioxidants such as coenzyme Q10 (CoQ10) declines leading to an increased presence of the visible signs of aging. CoQ10 is required for ATP synthesis in the mitochondria and also functions as an antioxidant in cell membranes, suggesting the existence of non-mitochondrial sites of synthesis. In the present study, we aimed at developing an approach to modulate the expression of Ubiad1, a prenyltransferase previously described in the Zebra fish, by using a synthetic peptide. Our results showed that Ubiad1 is expressed in the epidermis, mainly located in the Golgi apparatus. In addition, downregulation of Ubiad1 also exhibited H2O2-mediated ROS accumulation. Modulation of Ubiad1 protects keratinocytes submitted to oxidative stress from ROS accumulation and lipid peroxidation, and helps to preserve plasma membrane mechanical properties. Our results present a new strategy involving endogenous coenzyme Q10 synthesis by the modulation of non-mitochondrial Ubiad1 enzyme, to preserve skin appearance from visible aging signs and confirms that the CoQ10 existing in cell membranes is synthetized out from the canonical mitochondrial pathway.

The skin is a complex sensory system, reacting to the surrounding environment through an integrated network of cell receptors. Among them, G Protein-Coupled Receptors (GPCRs) represent a major way for cells to sense a large variety of environmental signals. Recent studies identified several extra-retinal opsins localized in human epidermal cells, three of them being sensitive to blue light (400-495 nm). As Light-Emitting Diode (LED)-based devices are increasingly part of our daily life, excessive exposure to artificial blue light can be considered as a source of indoor pollution, which has been shown to impact the circadian rhythm. The aim of the present study was to evaluate opsin expression in human skin. The localization of blue light-sensitive opsins was investigated in cultures of human keratinocytes and melanocytes and in human skin biopsies. Our results showed the expression and distribution of opsin 1 short-wavelength-sensitive, opsin 2 (Rhodopsin) and opsin 3 (Encephalopsin) within the epidermis, and their variation following exposure to blue light emitted from LEDs. In addition, our observations revealed a specific cellular localization of opsin 3 during mitosis. Taken together, these results emphasize the role of opsin photoreceptors in maintaining physiological conditions within the epidermis, and their implication in the skin’s response to stressful conditions induced by blue light.

OBJECTIVE:Human epidermis provides the body a barrier against environmental assaults. To assume this function, the epidermis needs the renewal of keratinocytes allowed by constant mitosis, which replace the exfoliating corneocytes. Keratinocyte stem cells (KSCs) located in the basal epidermis are mitotically active, self-renewing and govern the epithelial stratification by producing renewed source of keratinocytes. Protein complex such as the chromosomal passenger complex (CPC) allows the correct development of this process. The CPC is composed of four members: INCENP, survivin, borealin and aurora kinase B, and the disruption of the CPC during cell division induces mitotic spindle defects and improper repartition of chromosomes. The aim of our study was to investigate the implication of CRM1 and survivin in the progress of mitosis in skin keratinocytes. METHODS:Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched population of keratinocytes was isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin and CRM1 expression levels were assessed by immunofluorescence and immunoblotting. Specific siRNAs for each CPC member and for CRM1 were used to determine the relationship between these proteins. Survivin-specific siRNA was used to induce the apparition of mitotic abnormalities in cultured keratinocytes. RESULTS:We demonstrated the ability of our compound 'IV08.009' to modulate the expression level of survivin and CRM1 in keratinocytes and in skin biopsies. We observed that members of the CPC are interdependent: siRNA-induced inhibition of one component caused a decrease in the expression of all other CPC members. Downregulation of survivin or CRM1 induced mitotic abnormalities in keratinocytes. However, decreased number of mitotic abnormalities was observed in keratinocytes after 'IV08.009' application. CONCLUSION:Basal keratinocytes may divide frequently during skin lifespan, and signs of deterioration could appear such as loss of protein factors required for correct mitosis. Our findings suggest that mitotic abnormalities can be prevented by the modulation of CRM1 and survivin. We demonstrated the ability of compound 'IV08.009' to efficiently protect cultured keratinocytes from mitotic abnormalities.

Opsins that will convert it into electrical signals delivered to the brain. Besides this visual effect, this process also leads to non-visual effects including melatonin secretion, regulation of body temperature, mood, alertness and clock gene expression. The visible spectrum extends between 400 to 700 nm, and blue light (400-495 nm) represents the highest energy part of the visible light. In our everyday life, it appears that we are more and more exposed to light-emitting electronic devices containing much artificial blue light, which was shown recently to affect the circadian rhythms. In the skin, blue light has been used in many therapeutic approaches including inflammatory and hyperproliferative conditions such as psoriasis and acne. However, the role of blue light over-exposure on the physiological skin condition has been poorly described. Moreover, recent studies identified opsin receptor expression in human epidermal cells, 3 of them being sensitive to blue light. In the present study, we aimed at studying the effect of blue light exposure on Normal Human Keratinocytes (NHK) using LED irradiation. We determined exposure conditions generating ROS accumulation in NHK. In these conditions, we showed that the levels of circadian proteins PER1 and CRY1 were significantly disrupted after repeated blue LED irradiations. Moreover, we observed less opsin OPN1 SW, OPN2 and OPN3 expression at specific wavelengths of blue LED over-exposure. We also treated NHK with a vegetal extract and observed partial preservation of these markers from blue LED effects. Our studies showed that the expression of circadian proteins and opsin receptors could be disrupted by blue LED irradiation in normal human keratinocytes, associated with oxidative stress response. This study represents a new approach regarding skin sensitivity to blue light through opsin receptors.

In the visible spectrum, blue light (400-495 nm) represents the High Energy Visible (HEV) light associated with high photon energy. The majority of blue light comes from the sun, but it appears that we are more and more exposed to artificial light sources, including light-emitting electronic devices that emit significant amounts of blue light. Recently, opsin receptor expression has been reported in human epidermal cells, with three of them being sensitive to blue light. The role of blue light overexposure on the physiological skin condition has been poorly described. The aim of the present study was to evaluate the effect of blue light exposure on Normal Human Keratinocytes (NHK) using LED irradiation. Exposure conditions generating Reactive Oxygen Species (ROS) accumulation were first determined in NHKs. In these conditions, the levels of opsins OPN1 SW, OPN2 and OPN3 were observed to have decreased at specific wavelengths of blue LED. We also treated NHKs with a peptidic extract of Cocoa porcelana and observed a partial preservation of keratinocyte photoreceptors after blue LED exposure. Moreover, ex vivo studies on skin biopsies showed the effects of blue LED overexposure on the elastic fiber network and its preservation associated with the application of the extract. To evaluate the positive effect of a peptidic extract of Cocoa porcelana on the visible signs of aging linked to prolonged exposure to blue light, an in vivo study was performed on the faces of 20 volunteers. At the end of the treatment, wrinkle parameters were observed to have decreased significantly on the treated side compared to the placebo side. Taken together, our results showed that the expression of opsin receptors could be disrupted by blue LED overexposure in NHKs, associated with an oxidative stress response. Moreover, the in vivo study showed an association with an observed decrease in wrinkle appearance. These studies represent a new approach regarding skin sensitivity to blue light through opsin receptors.