How to help the skin cope with glycoxidation.

BACKGROUND: Protein glycation refers to the spontaneous reaction of reducing sugars with proteins and the subsequent formation of stable advanced glycation end products (AGEs). Glycation is linked with oxidative stress, and this association is called "glycoxidation". Glycoxidation alters the protein structure and function and causes tissue aging, as seen in human skin. Therefore, research on substances inhibiting glycoxidation appears to be crucial in the prevention of skin aging. With this aim, several plant extracts have been screened for antiglycation activity, and the results of the best candidates are presented in this article. METHODS: Glycation was studied on human skin proteins (collagen, elastin, and albumin) and on a model of reconstructed skin. Oxidative stress has been addressed by testing the copper-induced low-density lipoprotein oxidation, ultraviolet irradiation of glycated dermis, and carbonyl activation of human dermal fibroblasts. A clinical test evaluated the extent of oxidative stress induced by ultraviolet A irradiation. RESULTS: Among the tested products, several plant extracts have decreased the glycation effects on skin proteins collagen, elastin, and albumin. In addition, a plant extract has significantly inhibited the different forms of oxidative stress associated with protein glycation. CONCLUSIONS: We have demonstrated that plant extracts can relieve the deleterious effects of glycation on human skin. Moreover, a plant extract rich in antioxidant molecules has also significantly preserved the human skin from glycoxidation attacks.

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.

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.

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp.

Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22-70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (H2O2). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.

The Caucasian and African skin types differ morphologically and functionally in their dermal component.

In the literature, most reported differences between African and Caucasian skin properties concern pigmentation and barrier function of the stratum corneum and related photoprotective properties. However, little is known about differences in morphology and possibly related biological functions. In this study, we investigated: (i) architectural differences of Caucasian and African mammary skin biopsies using microscopy, (ii) comparative constitutive expression of cytokines, matrix metalloproteinase 1 (MMP-1) and its inhibitors in papillary dermal fibroblast (pF) and reticular dermal fibroblast (rF) cultures in order to reveal biological features. (i) Neither epidermis thickness nor superficial dermis thickness was significantly different in African versus Caucasian subjects. However, the dermal-epidermal junction (DEJ) length in African skin was about threefold that in Caucasian skin. No differences were noticed as regards elastic and collagen fibre organization. (ii) In papillary fibroblast cultures, a significantly higher level of monocyte chemotactic peptide-1 (MCP-1) protein was found in cell cultures from African donors when compared with that from Caucasians. With regard to keratinocyte growth factor (KGF), the ratio of papillary to reticular fibroblast expression was found to be twofold greater in cell cultures from African donors compared with that from Caucasian donors. The same trend was found regarding MMP-1 and tissue inhibitor metalloproteinase protein 1 (TIMP-1) protein expression. African skin displays a greater convolution of the DEJ and a higher papillary fibroblast activity. These findings reveal that differences between African and Caucasian skin do not only affect upper epidermis but also dermal functions and dermal-epidermal cellular interactions.

Ultraviolet-B irradiation induces epidermal up-regulation of heparanase expression and activity.

Heparan sulfate (HS) glycosaminoglycans are abundant components of basement membranes and cell surfaces where they are present associated with specific core-proteins to form proteoglycans, mainly perlecan, glypicans and syndecans. They play many roles such as modulation of cell proliferation and differentiation, cell-matrix adhesion and assembly. It was previously shown that HS content decreases during skin aging. This decrease could be explained either by a decrease of HS synthesis or by an increased activity of its degrading enzyme, heparanase (Hpse-1). Since UV-B irradiation is one of the most important factor for skin photo-damage, we decided to study the effects of UV-B irradiation on heparanase expression and activity in human epidermal keratinocytes. Normal human keratinocytes and reconstructed epidermis were submitted to increasing doses of UV-B. HPSE1 mRNA levels were measured using real time PCR and heparanase enzymatic activity was quantified in human keratinocyte cultures using a microtiter-based assay. Expression and distribution of Hpse-1 were also studied in reconstructed epidermis by immunofluorescence. Both HPSE1 mRNA level and heparanase enzymatic activity were increased after UV-B irradiation of keratinocyte cultures in a time and dose-dependent manner. Protein expression of Hpse-1 was also up-regulated with increasing doses of UV-B in reconstructed epidermis. Increase of Hpse-1 expression and activity in the epidermis after UV-B irradiation could contribute to skin photo-aging.

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation.

PURPOSE: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair. MATERIALS AND METHODS: The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA). RESULTS: SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not. CONCLUSIONS: FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2.

Ultraviolet-B irradiation induces differential regulations of hyaluronidase expression and activity in normal human keratinocytes.

Skin aging is a complex process determined by genetic factors (intrinsic aging) and environmental factors (extrinsic aging). One of the most influential environmental factor is UV-B irradiation. Hyaluronic acid (HA) is an abundant component of skin extracellular matrix where it plays many roles such as hydration and architectural support. Downregulation of HA during photoaging was reported previously. Changes in expression and function of its degrading enzymes, the hyaluronidases (Hyals) might be involved in this decrease. In the present study, normal human keratinocytes were submitted to increasing doses of UV-B. The mRNA expression of HYAL1, HYAL2 and HYAL3 and the hyaluronidase enzymatic activity were quantified using real-time PCR and a microtiter-based assay, respectively. After UV-B irradiation, HYAL1 mRNA expression was upregulated whereas HYAL2 and HYAL3 mRNAs were downregulated and hyaluronidase enzymatic activity was increased in both cell layer and culture medium. In parallel, immunohistochemical studies performed on UV-B irradiated reconstructed epidermis confirmed that Hyal-1, Hyal-2 and Hyal-3 protein expression were differently regulated by UV-B. Taken together, our results demonstrate that UV-B irradiation induces differential regulations of hyaluronidase expression and enzymatic activity in human keratinocytes. These differential modulations of hyaluronidase expression and activity by UV-B could contribute to cutaneous photoaging.

[Fibroblast subpopulations: a developmental approach of skin physiology and ageing]. / Etude des sous-populations de fibroblastes: une approche "développementaliste" de la physiologie et du vieillissement de la peau.

Skin is an organ whose function is far beyond a physical barrier between the inside and the outside of the body. Skin as the whole organism is subjected to ageing which concerns skin mostly in its dermal and deepest component which is also its matricial component. The dermis is a tissue rich in matricial elements and poor in cellular content and it is generally admitted that modifications occurring in the matrix are those which mostly contribute to skin ageing, by altering its biomechanical properties. Therefore it is common to address questions related to skin ageing by considering alterations in matrix molecules like collagen. Actually the dermis is a complex tissue both matricial and cellular and is divided between a superficial dermis close to epidermis and a deep dermis much thicker and histologically different. Several years ago we have undertaken investigations related to fibroblasts which are the cells responsible for the formation and maintenance of the dermis, aiming at isolation, culture and characterization of the fibroblasts from the superficial dermis also called papillary dermis and fibroblasts from the deep dermis also called reticular dermis. We were able to show that these fibroblasts in classical culture on plastic exhibit very different morphologies associated with different secretion properties and we have confirmed and expanded such observations revealing different phenotypes by incorporating these cells in reconstructed skin which allows the reproduction of a three-dimensional architecture recalling skin in vivo especially after grafting onto the nude mouse. We also raise the question of how these two dermal regions appear during the formation of the dermis and the question of their fate during ageing. Progress in solving these questions would certainly appear to be very useful for a better understanding of skin physiology and ageing and would hopefully provide new strategies in anti-ageing research.

Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging.

Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr) were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin.