COUNTERACTING AGING PHENOMENA BY NEW PURE TETRAPEPTIDES WITH TARGETED EFFICACY

Summary In the skin, proteoglycans are present both in the epidermjs and the dermis. In the latter layer, a group of small proteoglycans ( < 60 kDa) plays an important role in fibrillogenesis, growth factors modu lation and derma! homeostasis. The epidermis also synthesizes severa! small proteoglycans, in volved in keratinocyte functionality. The knowledge about the alteration of the synthesis and the structure of such small proteoglycans during skin aging used to be rather lirillted in the past. In fundamental studies, Laboratoires Sérobiologiques could recently identify two small proteoglycans whose influence in skin proper functioning is significant, and whose synthesis decreases with agi ng. Lumican is located in the dermjs, where it is invo lved in the formation of the collagen fibers and consequently in the skin firmness and thickness. Syndecan-l occurs preferentially in the supra-basai layers of the epidermis; it is jointly implicated in the epidermal cohesion. The dermo-epidermal junction assures the connection between dermis and epiderrills. With certain anchor molecules like collagen XVII, hemidesmosomes attach the basai cells to the basai membrane. The stimulation of the synthesis of lumican in derma! cells, of syndecan-l in epidermal cells and of collagen XVII in the basai layer counteracts the aging process on three different levels in the skin . In order to identify ingredients with a corresponding activity, skin specific DNA-arrays were used, allowing to selectively analyze the expression of genes highly implicated in the skin physiology. Two efficient acetylated tetrapeptides (respectively AcTPl and AcTP2) were selected. The efficacy of both peptides was further demonstrated on celi cultures, skin models and finally in clinica] studies. 63 Counteracting aging phenomena by new pure tetrapeptides with targeted efficacy Riassunto I proteoglicani sono presenti sia a livello dell'epidermide che del derma. Nel derma un gruppo di proteoglicani a basso peso molecolare (p.m) ( <60k Da) g ioca un ruolo importante nella fibri llo genesi, nella modulazione dei fattori di crescita e nell 'omeostasi di questo strato cellulare. Comunque, anche l'epidermide sintetizza d iversi piccoli proteoglicani, coinvolti nella fun zional ità dei cheratinociti. Negli anni passati l'alterazione che si verifica durante l' invecchiamento nella sintesi di stru tture quali i proteoglicani a basso p.m era poco conosciuta. Recentemente i laboratori della Seròbiologique hanno identifi cato due piccoli proteoglicani che influenzano in modo s ignificativo le funzioni cutanee, la cui sintesi viene a ridursi durante l' invecchiamento. Così il proteoglicano denominato Lumican è localizzato nel derma dove è coinvolto nella formazione delle fibre di collageno e quindi nello spessore e nella compattezza della cute. Il proteoglicano Syntecam-l s i riscontra normalmente negli strati soprabasali dell'epidermide ed è implicato nella coesione delle cellule epiderm iche. La g iunzione dermo-epidermica rappresenta il punto di congiunzio ne tra epidermide e derma attraverso l'atti vità di legame svolta da alcu ne molecole quali il collagene XVII e gli emodesmosomi . La stimolazione esercitata rispettivamente da Lumican a li vello delle cellule derm iche e del coll agene XVII esercita un effetto positi vo sul processo dell ' invecchiamento cutaneo a tre di versi li velli . Per verificare l'effettiva attività svolta dagli ingredienti in studio, ne è stata verificata l'efficacia con una serie di DNA specifici anali zzando seletti vamente l'espressione dei geni implicati a li vello fisiologico. Sono stati così selezionati due specifici tetrapeptidi acetilati (AcTPI e AcTP2). L'efficacia di entrambi i peptidi è stata d imostrata su culture cellulari , su modelli di cute ed infine a li vello cli nico. 6

The evolution and use of skin explants: potential and limitations for dermatological research.

International audienceDifferent models have been developed to understand the biology of skin or to test pharmaceutical/cosmetic products. These models can be in vitro models that possess advantages such as mono and co-culture models in 2D, which are very reproducible, or organotypic models (skin explant and reconstructed skin) that present a 3D organisation. Animal or human in vivo models allow studies that are closer to reality. In virtuo models developed on computers control all known parameters and do not require animals. The major limitations of these models are the lack of 3D structure for in vitro culture, the variability of results from organotypic models, ethical problems inherent to human and animal tests and the presence of numerous unknown parameters in in virtuo systems. Despite their limitations, skin explants seem to be an interesting model for studies. Skin explants may be kept from a few hours to 10-14 days on supports or directly in culture medium. These explants are generally cultivated at 37 °C, 5% CO(2), preferentially in serum-free conditions. Three basic techniques are used to characterise these models: histological stains, proliferation, apoptosis and cytotoxicity tests. Skin explants could be a very convenient model to study wound-healing, inflammation processes, autoimmune diseases, malignant transformation, stress, ageing, and to serve as screening tests

Effects of the re-innervation of organotypic skin explants on the epidermis.

International audienceThe nervous system takes part in skin homeostasis and interacts with skin cells. In in vitro organotypic skin models, these interactions are lost owing to the absence of nerve endings. We have developed an in vitro organotypic skin model based on a re-innervated human skin explant using primary sensory neurons from the dorsal root ganglia of rats. After 10 days of co-culture between skin explant and neurons, a dense network of nerve fibres was observed. The epidermis and dermis presented nerve fibres associated with cellular body from sensory neurons introduced in the co-culture. Epidermal thickness, cell density and quality of re-innervated skin explant were all higher when skin explants were re-innervated by sensory neurons at 10 days of culture. Proliferation of epidermal cell was not modified, but the apoptosis was significantly diminished. Hence, this innovative model of co-cultured skin explants and neurons allows better epidermal integrity and could be useful for studies concerning interactions between the skin and its peripheral nervous system

Hair Histology and Glycosaminoglycans Distribution Probed by Infrared Spectral Imaging: Focus on Heparan Sulfate Proteoglycan and Glypican-1 during Hair Growth Cycle

The expression of glypicans in different hair follicle (HF) compartments and their potential roles during hair shaft growth are still poorly understood. Heparan sulfate proteoglycan (HSPG) distribution in HFs is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this report, a novel approach is proposed to assess hair histology and HSPG distribution changes in HFs at different phases of the hair growth cycle using infrared spectral imaging (IRSI). The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. The findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs, and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia

Effect of human skin explants on the neurite growth of the PC12 cell line.

International audienceThe skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co-cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells co-cultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells

Characterization of neurons from adult human skin-derived precursors in serum-free medium : a PCR array and immunocytological analysis.

International audienceAdult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum-free medium, stem cells from abdominal skin were cultured in serum-free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil-red-O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki-67 for proliferative cells, (v) FM1-43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1-43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown

An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential