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.

The Glypican-1/HGF/C-Met and Glypican-1/VEGF/VEGFR2 Ternary Complexes Regulate Hair Follicle Angiogenesis.

The hair renewal involves changes in the morphology of the hair follicle and its micro-vascularization. In alopecia, the hair cycle is accelerated, resulting in the formation of thinner and shorter hair. In addition, alopecia is associated with a decrease in the micro-vascularization of the hair follicles. In this study, the role of glypicans (GPCs) was analyzed in the regulation of the angiogenesis of human dermal microvascular endothelial cells (HDMEC). The analysis of glypican gene expression showed that GPC1 is the major glypican expressed by human keratinocytes of outer root sheath (KORS), human hair follicle dermal papilla cells (HHFDPC) and HDMEC. KORS were demonstrated to secrete VEGF and HGF. The HDMEC pseudotube formation was induced by KORS conditioned media (KORSCM). It was totally abrogated after GPC1 siRNA transfection of HDMEC. Moreover, when cleaved by phospholipase C (PLC), GPC1 promotes the proliferation of HDMEC. Finally, GPC1 was shown to interact directly with VEGFR2 or c-Met to regulate angiogenesis induced by the activation of these receptors. Altogether, these results showed that GPC1 is a key regulator of microvascular endothelial cell angiogenesis induced by VEGF and HGF secreted by KORS. Thus, GPC1 might constitute an interesting target to tackle alopecia in dermatology research.

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.

Development of new 3D human ex vivo models to study sebaceous gland lipid metabolism and modulations.

OBJECTIVES: Sebaceous glands maintain skin homeostasis by producing sebum. Low production can induce hair loss and fragile skin. Overproduction provokes seborrhoea and may lead to acne and inflammatory events. To better study sebaceous gland maintenance, sebocyte maturation, lipid production and ageing or inflammatory processes, we developed innovative 3D ex vivo models for human sebaceous glands. MATERIALS AND METHODS: Culture conditions and analytical methods optimized on sebocyte monolayers were validated on extracted sebaceous glands and allowed the development of two 3D models: (a) "air-liquid" interface and (b) human fibronectin-coated "sandwich" method. Lipid production was assessed with microscopy, fluorometry or flow cytometry analysis after Nile Red staining. Specific lipids (particularly squalene and peroxidized squalene) were measured by Gas or liquid Chromatography and Mass spectrometry. RESULTS: This study allowed us to select appropriate conditions and design Seb4Gln culture medium inducing sebocyte proliferation and neutral lipid production. The "air-liquid" model was appropriate to induce sebocyte isolation. The "sandwich" model enabled sebaceous gland maintenance up to 42 days. A treatment with Insulin Growth Factor-1 allowed validation of the model as we succeeded in mimicking dynamic lipid overproduction. CONCLUSION: Functional sebocyte maturation and physiological maintenance were preserved up to 6 weeks in our models. Associated with functional assays, they provide a powerful platform to mimic physiological skin lipid metabolism and to screen for active ingredients modulating sebum production.

Understanding Solar Skin Elastosis-Cause and Treatment.

Photoageing, also called actinic ageing, is the main cause of prematurely aged skin. Our expertise in elastic fibers has led us to discover a process triggered in response to ultraviolet (UV) light and which upsets the balance of elastin fibers: there is too much elastin and insufficient lysyl oxidase (LOXL1) enzyme to form functional elastic fibers. This imbalance then leads to an accumulation of nonfunctional elastin, which forms aggregates. In addition to this imbalance, UV rays also induce elafin synthesis by fibroblasts. Known to be a marker of elastotic aggregates, elafin crystallizes the elastin fibers and stimulates the formation of aggregates that cannot be naturally eliminated by the skin. We developed a Hamamelis virginiana leaf extract that was able to restore both the balance between elastin and LOXL1 and to decrease the elafin synthesis to fight and correct the damage. This specific Hamamelis virginiana extract increased LOXL1 expression by twofold and decreased elafin synthesis. As a consequence, elastic fibers became functional and aggregates of unfunctional fibers decreased. The specific Hamamelis extract activity was confirmed in vivo with decreasing wrinkles and improving skin firmness.

O2 level controls hematopoietic circulating progenitor cells differentiation into endothelial or smooth muscle cells.

BACKGROUND: Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs) or contractile smooth muscle cells (SMCs) while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS: Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2) or normoxic (21% O2) environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks) showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd) passage) in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs) signature of phenotype stability. CONCLUSION/SIGNIFICANCE: We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions. These aspects are of the highest importance for tissue engineering strategies. These results highlight also the determinant role of the tissue environment in the differentiation process of vascular progenitor cells.

Small vessel replacement by human umbilical arteries with polyelectrolyte film-treated arteries: in vivo behavior.

OBJECTIVE: The aim of this study was to evaluate the patency of human umbilical arteries treated with polyelectrolyte multilayers (PEMs) after rabbit implantation. BACKGROUND: The development of small-caliber vascular substitutes with high patency after implantation remains a real challenge for vascular tissue engineering. METHODS: Cryopreserved human umbilical arteries were enzymatically de-endothelialized and the luminal surfaces were coated with poly(styrene sulfonate)/poly(allylamine hydrochloride) (PSS/PAH) multilayers. The PEM-untreated arteries and PEM-treated rabbit carotids were used as graft control. The native rabbit carotids were bypassed by grafts. RESULTS: The Doppler ultrasound evaluation, performed in vivo, showed that all PEM-treated grafts remained patent during the full experimental period, whereas after only 1 week, no blood circulation was detected in untreated arteries. Scanning electron microscopy and histological graft examination showed pervasive thrombus formation on the luminal surface of untreated arteries after 1 week and clean luminal surface for treated arteries for at least up to 12 weeks. The arterial wall cells were identified through alpha-smooth muscle actin alphaupsilondelta platelet endothelial cell adhesion molecule-1 expression. The smooth muscle cells positive to alpha-smooth muscle actin were identified in adventitia and media and the endothelial cells positive to platelet endothelial cell adhesion molecule in intima. Von Kossa reaction didn't reveal any calcium salt deposits on the wall arteries, suggesting a good wall remodelling with no sign of graft rejection. CONCLUSIONS: The in vivo evaluation of human umbilical arteries treated with PSS/PAH multilayers demonstrated a high graft patency after 3 months of implantation. Such modified arteries could constitute a useful option for small vascular replacement.

Polyelectrolyte Films Boost Progenitor Cell Differentiation into Endothelium-like Monolayers.

Rapid differentiation of endothelial progenitor cells (EPCs) into confluent mature endothelial cells is important in tissue engineering for the design of autologous, nonthrombotic, vascular grafts. A new method based on EPC culture on poly(sodium-4- styrene-sulfonate)/poly(allylamine hydrochloride), that is, polyelectrolyte-multilayer-coated substrates, reduces the time from two months to two weeks.

The ideal small arterial substitute: Role of cell seeding and tissue engineering.

Although autogenous vessels are useful in surgery, often patients cannot furnish suitable vessels. If there are not available, two possible alternatives for vessel replacements are to use vascular synthetic prostheses such as Dacron((R)) and polytetrafluoroethylene (PTFE) or cryopreserved allografts. However, their success has been limited to replace small-diameter (<6 mm) arterial vessel because of their high thrombogenicity and compliance mismatch. On account of a clear clinical need for a functional arterial substitute, tissue engineering techniques have been developed. This review encompasses the use of mature endothelial, endothelial progenitor and bone marrow cells combined with natural or synthetic scaffolds whose surface has been modified with multiple origin matrices.