Louis Danoux

Université de Bretagne Occidentale, Brest, Brittany, France

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Publications (18)34.33 Total impact

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    ABSTRACT: Abstract 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.
    Clinical Chemistry and Laboratory Medicine 04/2013; · 3.01 Impact Factor
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    ABSTRACT: The 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.
    Experimental Dermatology 03/2013; 22(3):224-225. · 3.58 Impact Factor
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    ABSTRACT: Adult 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.
    Experimental Dermatology 03/2012; 21(3):195-200. · 3.58 Impact Factor
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    ABSTRACT: The 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.
    Experimental Dermatology 02/2012; 21(2):156-8. · 3.58 Impact Factor
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    ABSTRACT: 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.
    Journal of photochemistry and photobiology. B, Biology 10/2011; 106:107-12. · 3.11 Impact Factor
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    ABSTRACT: 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.
    Photochemistry and Photobiology 06/2011; 87(5):1105-12. · 2.29 Impact Factor
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    ABSTRACT: We have previously shown that axonal growth from a subset of sensory neurons was promoted by keratinocytes when the two cell types were co-cultured in a low calcium medium. This phenomenon involves the production of one or several diffusible factors. Here we show that the neuritogenic effect of keratinocytes was significantly reduced in the case of rat primary sensory dorsal root ganglion (DRG) neurons, or completely suppressed in the case of the sensory neuron cell line ND7-23, when the activity of neurotrophin receptors (Trk receptors) was blocked with K252a. This trophic effect apparently involved the activation of tyrosine kinase receptors A and B (TrkA and TrkB) expressed by subpopulations of small- to medium-sized DRG neurons, or only of TrkA receptors in the case of ND7-23 neurons. A residual neurite growth promoting effect of keratinocytes persisted in a fraction of DRG neurons after Trk receptor blockade. This effect was mimicked by the steroid dehydroepiandrosterone (DHEA) but not by other steroids such as pregnenolone, progesterone or 17beta-estradiol. The use of pharmacological agents which inhibit different steps of steroidogenesis indicated that DHEA was probably synthesized from cholesterol in keratinocytes. Our results strongly suggest that DHEA might act as a neurotrophic signal derived from keratinocytes to promote axonal outgrowth from a subpopulation of sensory neurons.
    Neuroscience 04/2009; 159(2):514-25. · 3.12 Impact Factor
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    ABSTRACT: IFSCC Magazines, 12 (2009) (1) 25–30 Approximately 40% of the population (all skin categories and phototypes) complain of sensitive skin. Sensitive skin is healthy but overresponsive, meaning it reacts faster and more intensely to several parameters including environmental factors such as temperature changes and the sun, use of cosmetic products, and certain medicines. It experiences discomfort, tingling, burning and intolerance to certain types of products, a condition referred to as neurosensitivity characterized by a lower threshold of tolerance. Currently, all of the causes are not known but an increase in the permeability of the stratum corneum and an exaggeration of the nerve response are considered to be involved in the phenomenon of sensitive skin. Lifestyle factors including tobacco, alcohol, stress, fatigue and emotions also have an effect. A new synthetic tetrapeptide, N-acetyl-L-tyrosyl-L-prolyl-L-phenylalanyl-L-phenylalaninamide (Ac-YPFF-NH2), mimicking a natural opioid peptide was developed with the aim to decrease skin nerve ending stimulation. This tetrapeptide was demonstrated in vitro to reduce cutaneous overreactivity by decreasing release of calcitonin gene-related peptide from sensory neurons via an agonist effect on the μ opioid receptors and in vivo to improve the comfort of sensitive skin by decreasing unpleasant sensations and pain induced by heat and capsaicin. This tetrapeptide targeting an exaggerated nerve response helps to relieve sensitive skin by normalizing the tolerance threshold for environmental factors or certain topically applied uncomfortable products or skincare treatments. Keywords: Calcitonin gene-related peptide, μ opioid receptor, nerve endings, peptide, sensitive skin
    International Journal of Cosmetic Science 01/2009; 31(6):480-480.
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    ABSTRACT: Le tissu adipeux est un organe jouant un rôle majeur dans le maintien de l’homéostasie métabolique. De plus, la masse grasse a acquis le statut d’organe endocrine. Enfin, récemment, la présence de cellules du système immunitaire a été mise en évidence dans le tissu adipeux et suggérée comme étant responsable du développement de l’inflammation chronique de bas niveau associée à l’obésité. Ces échanges de métabolites, d’hormones et de cellules entre compartiment adipeux et sanguin démontrent le rôle prépondérant de l’endothélium dans la masse grasse. Or, chez l’homme aucune étude n’a décrit l’endothélium du tissu adipeux. Cette étude a été menée afin de caractériser le système capillaire du tissu adipeux humain sous-cutané abdominal et d’évaluer l’influence du degré d’adiposité des patients sur l’état d’activation des cellules endothéliales du tissu adipeux humain. Nous avons déterminé, par observation in situ en immunofluorescence que le tissu adipeux humain possédait un réseau capillaire très développé caractérisé par la double expression des marqueurs de surface CD31 et CD34. L’analyse en cytométrie en flux du nombre de cellules CD31+/CD34+ du tissu adipeux de patientes normopondérées (indice de masse corporelle (IMC) compris entre 20 et 25) et obèses (IMC supérieur à 30) a montré que le nombre de cellules CD31+/CD34+ est maintenu constant, démontrant que la croissance excessive du tissu adipeux s’accompagne d’une extension de son réseau capillaire. La caractérisation par PCR quantitative en temps réel de transcrits exprimés dans les cellules endothéliales capillaires CD31+/CD34+ du tissu adipeux humain isolées par une technique d’immunosélection/déplétion a mis en évidence un état d’activation inflammatoire et angiogénique de ces cellules chez les sujets obèses. De plus, nous avons développé une technique de culture primaire de ces cellules permettant le maintien de l’expression des marqueurs endothéliaux. En conclusion, cette étude a permis de mettre au point des techniques d’immunohistochimie en fluorescence des réseaux capillaires du tissu adipeux humain et de culture primaire des cellules endothéliales. De plus, nos résultats montrent que le développement de la masse grasse chez l’homme module le phénotype des cellules endothéliales (activation inflammatoire et angiogénique).
    Archives of Cardiovascular Diseases - ARCH CARDIOVASC DIS. 01/2009; 102.
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    ABSTRACT: IFSCC Magazine, 11 (2008) (1) 21–29 Proteoglycans take an important part in tissue homeostasis. In the skin, Proteoglycans are present in the extracellular matrix of dermis, particularly with lumican which plays an important role in dermal homeostasis. In the epidermis, several small proteoglycans such as the syndecans are synthesized and play an important role in keratinocyte activation. There is much data on the alteration during skin aging of the synthesis and structure of glycosaminoglycans and some proteoglycans but little information on small proteoglycans, in particular lumican and syndecan-1.We recently observed a decrease in the synthesis of these two small proteoglycans with aging. We confirm in this work the decrease in lumican in dermis and syndecan-1 in epidermis with aging. These proteoglycans represent original important targets for cosmetology in the fight against skin aging. In different in vitro models, two synthetic acetyl tetrapeptides, AcTP1 and AcTP2, stimulate the synthesis of lumican and syndecan-1, respectively. The beneficial action of AcTP1 on skin thickness and firmness and of AcTP2 on epidermal cohesion has been confirmed in vivo.
    International Journal of Cosmetic Science 01/2009; 31(2):154-154.
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    ABSTRACT: The epidermis, the outermost structure of the skin, fulfils important roles as a physical barrier between the organism and its environment and as a neuroendocrine, immune and sensory organ. It is innervated by unmyelinated sensory fibres conveying nociceptive and thermoceptive information. Little is known concerning the functional interactions between these sensory fibres and the keratinocytes, which constitute 95% of the epidermal cells. We have developed a coculture model of primary rat sensory neurons and keratinocytes, as well as of equivalent cell-lines: ND7-23 neurons and A431 keratinocytes. We show that primary dorsal root ganglion neurons survive well in a standard keratinocyte reference medium containing a low concentration of calcium, but fail to extend axons. However, when neurons are cocultured with keratinocytes, axonal outgrowth is strongly stimulated. The use of a Transwell culture system indicated that the stimulation of axonal growth depends on a soluble factor secreted by keratinocytes. Axon outgrowth was also induced by nerve growth factor or brain-derived neurotrophic factor, but not by neurotrophin 3 or glial cell-derived neurotrophic factor. Neurons cocultured with keratinocytes did not change their responses to ATP, capsaicin or high potassium solution, as measured by calcium imaging. The trophic effect of keratinocytes concerned essentially a population of medium-sized (17-25 microm) neurons, some of which expressed substance P-like immunoreactivity and responded to capsaicin. Our preparation, in which cells are maintained at low external calcium concentration, could represent a useful in vitro model for characterizing the effect of skin-derived guidance and trophic factors.
    European Journal of Neuroscience 08/2007; 26(1):113-25. · 3.75 Impact Factor
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    ABSTRACT: Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied. In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [3H]-glucosamine and [35S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor's age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor's age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin. Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.
    Molecular and Cellular Biochemistry 10/2005; 277(1-2):63-72. · 2.33 Impact Factor
  • C Jeanmaire, L Danoux, G Pauly
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    ABSTRACT: Non-enzymatic glycation occurring in normal human skin plays an important part in ageing. OBJECTIVES To visualize and quantify, in human subjects, the extent of glycation during human dermal intrinsic and actinic ageing, and to develop a reliable reproducible in vitro model for evaluating the efficacy of potential inhibitors of glycation. By immunohistochemistry using a monoclonal antibody recognizing carboxymethyl lysine, an advanced glycation end-product (AGE) (first objective), and by incubating dead de-epidermized dermis (DED) with glucose to simulate ageing-induced glycation in a human dermal equivalent model (second objective). We found that glycation of the dermis generally arises after 35 years, then increases rapidly with intrinsic ageing. We also noticed an enhancement of glycation by solar irradiation that occurred via glycation of the elastic fibre network or solar elastosis tissue. In the model, production of AGEs appeared in a time-dependent way, mimicking glycation observed in vivo during chronological ageing. Irradiation of DED before incubation with glucose strongly enhanced induction of AGEs, corresponding to the effect of solar irradiation on AGEs observed in vivo. These results confirm a marked increase of AGEs during intrinsic ageing in normal human skin and also suggest that glycation is enhanced in photoaged skin.
    British Journal of Dermatology 08/2001; 145(1):10-8. · 3.76 Impact Factor
  • Journal of cosmetic science 55(2):224-5. · 0.28 Impact Factor
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    ABSTRACT: Different 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.
    European journal of dermatology: EJD 20(6):671-84. · 1.95 Impact Factor
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    ABSTRACT: Learning from Nature: Biomimicry
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