A combination of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol stabilizes hypoxia-inducible factor 1-alpha and improves hair density in female volunteers.

OBJECTIVE: The aim of this study was first to demonstrate that a combination of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol could synergize in vitro on biological pathways associated with hair growth and then to demonstrate the benefit on hair density in a clinical study. METHODS: The effects of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol directly on the hypoxic inducible factor-1α protein (HIF-1α) and related genes expression were demonstrated on keratinocytes in culture in vitro using western-blot analysis and real time quantitative polymerase chain reaction analysis. The effect of resveratrol against oxidative stress induced by hydrogen peroxide treatment was studied in hair follicle and hair matrix cells in vitro using the sensitive probe Dichloro-dihydro-fluorescein diacetate (DCFH-DA). Finally, a randomized clinical study on hair density was conducted on 79 Caucasian female subjects to assess the effect of this combination of actives. RESULTS: Pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol stabilized HIF-1a protein and increased the expression of HIF-1α target genes. Resveratrol significantly reduced the oxygen peroxide-induced oxidative stress generated in hair follicle and hair matrix cells. The clinical study showed that a topical treatment with the combination significantly increased the hair density on women from 1.5 months. CONCLUSION: In addition to the antioxidant properties of resveratrol, the association of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol revealed a synergistic effect on the HIF-1α pathway. The results of the clinical study confirmed the importance of such a combination to increase the hair density.

L'alopécie peut affecter 50% des femmes au cours de leur vie ce qui induit une perte de leur estime de soi et une diminution de leur qualité de vie. Au-delà des solutions chirurgicales et des traitements pouvant induire des effets secondaires potentiellement dangereux, il y a un besoin d'améliorer l'efficacité des produits cosmétiques qui permettent de prévenir la chute des cheveux tout en préservant la sécurité des patients. Ainsi, nous avons sélectionné une combinaison de pyridine-2, 4-dicarboxylic acide diethyle ester et de resvératrol pour activer des voies biologiques associées à la croissance du cheveu. Nous avons d'abord montré, in vitro, que la combinaison de pyridine-2, 4-dicarboxylic acide diethyle ester et de resvératrol permet de stabiliser la protéine HIF-1α conduisant ainsi à un effet synergique sur l'expression de gènes clés de la voie HIF-1α. Nous avons aussi démontré, in vitro, que le resvératrol permet de protéger significativement les follicules pileux et les cellules de la matrice du stress oxydatif induit par traitement au peroxide d'hydrogène. En final, une étude clinique randomisée mesurant la densité capillaire a été réalisée sur 79 femmes caucasiennes. Cette étude montre qu'une application topique d'une solution contenant de 5% pyridine-2, 4-dicarboxylic acide diethyle ester et 0.25% de resvératrol augmentent significativement la densité capillaire chez les femmes après 1.5 mois. En conclusion, ces résultats démontrent l'intérêt de stimuler la voie HIF-1α tout en protégeant les cheveux et le scalp du stress oxydatif afin d'améliorer la croissance des cheveux chez les femmes.

Polygonum multiflorum Radix extract protects human foreskin melanocytes from oxidative stress in vitro and potentiates hair follicle pigmentation ex vivo.

OBJECTIVE: To examine the ability of an extract from traditional Chinese medicine, Polygonum multiflorum Radix, to protect melanocyte viability from oxidative stress, a key mechanism in the initiation and progression of hair greying. METHODS: To assess the antioxidant capacity of Polygonum multiflorum Radix extract, primary human foreskin melanocytes were treated with a commercially available Polygonum multiflorum Radix extract added to culture medium and exposed to hydrogen peroxide (H2 O2 ), using intracellular reactive oxygen species concentrations and glutathione/protein ratios as endpoints. To improve solubility for cosmetic uses, a new Polygonum multiflorum Radix extract was derived. As hair greying is the consequence of melanocyte disappearance in an oxidative stress environment, we checked whether the antioxidant capacity of the new Polygonum multiflorum Radix extract could preserve melanocyte viability in response to H2 O2 -induced oxidative stress, and preserve pigmentation within ex vivo human hair follicles. RESULTS: In vitro treatment of primary human foreskin melanocytes with traditional available Polygonum multiflorum Radix extract resulted in decreased intracellular ROS accumulation in response to H2 O2 exposure with a concomitant preservation of glutathione-to-protein ratio, consistent with a protective response against H2 O2 exposure and demonstrating the promise of this extract for protecting melanocytes against oxidative stress. Melanocytes treated with the improved Polygonum multiflorum Radix extract exhibited attenuated H2 O2 -induced cell death, demonstrating a clear cytoprotective effect. Treatment of ex vivo human hair follicles with the improved Polygonum multiflorum Radix extract resulted in a higher level of melanin compared to vehicle-treated controls, demonstrating an ex vivo protective effect on hair pigmentation. CONCLUSION: Polygonum multiflorum Radix extract protects in vitro primary human foreskin melanocytes from the deleterious effects of H2 O2 exposure and improves pigmentation within ex vivo human hair follicles, demonstrating the utility of Polygonum multiflorum Radix extract as a potential active ingredient for the protection of melanocytes against premature death. This data provides in vitro mechanistic evidence consistent with existing in vivo studies for the use of Polygonum multiflorum Radix extract as a strategy for the prevention of oxidative stress-induced hair greying, in line with traditional Polygonum multiflorum Radix uses.

Age-dependent changes in eumelanin composition in hairs of various ethnic origins.

Hair pigmentation is one of the most conspicuous phenotypes of humans. From a chemical point of view, however, data remain scarce regarding human hair pigmentation characteristics. To determine melanin content and composition in human eumelanic hair from individuals of different ethnic origins and at different ages, we collected hair from 56 subjects with eumelanic hair from each group of African-American, East Asian, and Caucasian origin. The 56 subjects consist of 14, seven each of males and females, each from four age classes of younger than 11, between 12 and 19, between 20 and 45, and older than 46. We analysed hair colour scale, total melanin value, and contents of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA). We measured age-dependent increases in the relative quantity of eumelanin in pigmented human hairs in the three ethnic groups. Regarding melanin composition, we observed an increase in the PDCA/PTCA ratio with age in African-American and Caucasian hairs until approaching the quite constant level of the ratio in East Asian hairs in the elderly individuals. Our results evidence differences in the content and composition of eumelanin in human hair among African-American, Caucasian and East Asian individuals. Furthermore, we show evidence of age-dependent changes in the quantity and quality of eumelanin in pigmented human hairs. In particular, the age-dependent modification of the PDCA/PTCA ratio, a marker for 5,6-dihydroxyindole units in eumelanin, suggests a chronological evolution of hair follicle melanocyte phenotype (e.g. decrease in dopachrome tautomerase expression).

TRP-2 expression protects HEK cells from dopamine- and hydroquinone-induced toxicity.

We previously reported that melanogenic enzyme TRP-2 (or DCT for DOPAchrome tautomerase) expression in WM35 melanoma cells resulted in increased intracellular GSH levels, reduction in DNA damage induced by free radicals, and decreased cell sensitivity to oxidative stress. These effects seemed to depend on a particular cellular context, because none of them were found to occur in HEK epithelial cells. We postulated that the TRP-2 beneficial effect observed in WM35 cells in the oxidative stress situation may relate to quinone metabolization and, more precisely, to the ability of TRP-2 to clear off related toxic metabolites, resulting in a global redox status modification. Here, a comparative protein expression profiling of catecholamine biosynthesis enzymes and detoxification enzymes was conducted in WM35 melanoma cells and in HEK epithelial cells, in comparison with normal human melanocytes. Results showed that WM35 cells, but not HEK cells, expressed enzymes involved in catecholamine biosynthesis, suggesting that their quinone-related toxic metabolites were present in WM35 cells but not in HEK cells. To address the issue of a possible TRP-2 beneficial effect toward quinone toxicity, cell survival experiments were then conducted in HEK cells using dopamine and hydroquinone at toxic concentrations. We showed that TRP-2 expression significantly reduced HEK cell sensitivity to both compounds. This beneficial property of TRP-2 was likely to depend on the integrity of its DOPAchrome tautomerase catalytic site, because both TRP-2(R194Q) and TRP-2(H189G), which have lost their DOPAchrome tautomerase activity, failed to modify the HEK cell response to dopamine and hydroquinone. These results suggest that TRP-2 acts on quinone metabolites other than DOPAchrome, e.g., in the catecholamine pathway, and limits their deleterious effects.

Human hair greying is linked to a specific depletion of hair follicle melanocytes affecting both the bulb and the outer root sheath.

BACKGROUND: Although hair greying is a very common phenomenon characterized by loss of pigment in the hair shaft, the events that cause and control natural hair whitening with age in humans are still unclear. OBJECTIVES: To decipher the origin of natural hair whitening. METHODS: Human hair melanocytes were immunohistochemically characterized at different stages of whitening. RESULTS: Loss of hair shaft melanin was found to be associated with a decrease in both bulb melanin content and bulb melanocyte population. Although few melanocytes were present in the bulbs of grey hair, they still expressed tyrosinase and tyrosinase-related protein-1, synthesized and transferred melanins to cortical keratinocytes as seen by the presence of melanin granules. In white hair bulbs, no melanocytes could be detected either with pMel-17 or vimentin labelling. Pigmented hair follicles are known to contain inactive melanocytes in the outer root sheath (ORS), and grey and white hairs were also found to contain some of these quiescent melanocytes. However, their population was decreased compared with pigmented hair follicles, ranging from small to nil. This depletion of melanocytes in the different areas of white hairs was detected throughout the hair cycle, namely at telogen and early anagen stages. In contrast, the infundibulum and sebaceous gland of both pigmented and white hairs showed a similar distribution of melanocytes. Furthermore, other distinct cell populations located in the ORS, namely putative stem cells, Merkel cells and Langerhans cells were equivalently identified in pigmented and white hairs. CONCLUSIONS: Thus, hair greying appears to be a consequence of an overall and specific depletion of bulb and ORS melanocytes of human hair.

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study.

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.

The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?

Up to now, the localization of stem cells in human anagen hair follicle relied on three complementary approaches; namely, detection of slow cycling cells, detection of high colony forming cells, and differential immunohistochemical staining. These techniques, however, gave conflicting results since stem cells were localized either as long label retaining cells in the so-called bulge area or as high colony forming cells in the lower third of the follicle. In the present study we investigated the expression of cytokeratin 19, a marker for putative stem cell-containing epithelial compartments, in order to characterize stem cell distribution in the human hair follicle throughout the hair cycle. We found that anagen human hair follicles contain two distinct reservoirs for stem cells located in the upper and lower thirds of the follicle. These two reservoirs fuse during the catagentelogen transition phase and individualize again in the newly forming anagen hair follicle.

Immunohistochemical analysis of tissue remodelling during the anagen-catagen transition of the human hair follicle.

The transition from the growth phase (anagen) to the involution phase (catagen) involves profound morphological changes in the human hair follicle. Club hair and epithelial column formation, for example, are key features of the catagen phase, which result in the disruption of physical interaction between the bulb and the dermal papilla. However, the dynamics and tissue remodelling that occur during this involution process remain largely unknown. Using monoclonal antibodies directed against K14 keratin, trichohyalin, transglutaminase I, desmoglein and Ki67 antigen, we followed the movements of each of the main hair follicle compartments during the onset of catagen. Our results indicate that the inner root sheath is an early target in this process, suggesting a key role for this compartment in the maintenance of hair follicle homeostasis.

The distribution of alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins identifies distinct subpopulations of basal keratinocytes in the outer root sheath of the human anagen hair follicle.

The human hair follicle is composed of different concentric compartments, which reflect different programmes of differentiation. Using monoclonal antibodies against alpha 2 beta 1 and alpha 3 beta 1 integrins we demonstrated a shift in their expression, from a basolateral distribution in the basal cells of the lower outer root sheath, to an apicolateral expression in the upper outer root sheath, as in epidermis. This shift takes place in a transition zone, localized to the midpart of the follicle. The distinct basolateral distribution of alpha 2 beta 1 and alpha 3 beta 1 integrins in the lower portion of the outer root sheath coincides with the presence of basal cell protrusions and is probably linked to the presence of the vitreous membrane which surrounds the bottom part of the anagen human hair follicle. Moreover, we showed that the expression of alpha 6 beta 4 integrin is discontinuous along the hair follicle and coincides with that of laminin 5. Together these results establish that within a given compartment-namely the outer root sheath-several domains can be clearly identified, which probably reflect the onset of successive differentiation pathways along the hair follicle.

Activation of cytoprotective prostaglandin synthase-1 by minoxidil as a possible explanation for its hair growth-stimulating effect.

Data from the literature indicate that nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, naproxen, piroxicam, or ibuprofen, induce hair loss in vivo. These NSAIDs are well-known inhibitors of both the cytoprotective isoform of prostaglandin endoperoxide synthase-1 (PGHS-1) and of the inducible form (PGHS-2). By immunohistochemical staining, we found that PGHS-1 is the main isoform present in the dermal papilla from normal human hair follicle (either anagen or catagen), whereas PGHS-2 was only faintly and exclusively expressed in anagen dermal papilla. Thus, PGHS-1 might be the primary target of the hair growth-inhibitory effects of NSAIDs. We thus speculated that activation of PGHS-1 might be a mechanism by which minoxidil (2,4-diamino-6-piperidinopyrimidine-3-oxyde) stimulates hair growth in vivo. We demonstrate here that minoxidil is a potent activator of purified PGHS-1 (AC50 = 80 microM), as assayed by oxygen consumption and PGE2 production. This activation was also evidenced by increased PGE2 production by BALB/c 3T3 fibroblasts and by human dermal papilla fibroblasts in culture. Our findings suggest that minoxidil and its derivatives may have a cytoprotective activity in vivo and that more potent second-generation hair growth-promoting drugs might be designed, based on this mechanism.