Induction of the skin endogenous protective mitochondrial MnSOD by Vitreoscilla filiformis extract.

Vitreoscilla filiformis (Vf), a filamentous bacteria living in fresh water is thought to contribute to the observed beneficial effects of Spa water on skin. An active fraction obtained from a Vf biomass was evaluated for its ability to modulate mRNA expression in cultured skin cells. cDNA array analysis was conducted first using a customized membrane including 1176 selected and fully identified genes involved in skin physiology and homeostasis then the newly developed full genome U133 plus 2.0 GeneChip from Affymetrix. The mitochondrial protective manganese superoxide dismutase (MnSOD/SOD-2) was identified as a preferentially induced mRNA target in both normal human dermal fibroblasts and keratinocytes. Induction at the transcriptional level in both cell types was confirmed using quantitative real time/polymerase chain reaction and a kinetic analysis revealed a maximal increase in mRNA expression 20 h after stimulation with Vf extract (Vfe). Using immunofluorescent (fluorescent cell sorter) analysis, an induction of MnSOD protein in both normal human dermal skin fibroblasts (x1.6; P < 0.01) and epidermal keratinocytes (x1.4; P < 0.01) was confirmed. As MnSOD is a major inducible free-radical scavenger in skin, these results suggest that the Vfe could induce skin cells to produce their own endogenous protective defences in vivo against both exogenous environmental stressors such as UV irradiation or microflora as well as to combat endogenous sources of deleterious free radicals involved in skin ageing. Finally, in order to confirm the in vivo potential of this original extract in human, we evaluated its protective activity vs. placebo on the generation of sunburn cells in epidermis under UVB stress. As expected from in vitro profiling, Vfe was indeed found to significantly inhibit the appearance of sunburn cells in UVB-exposed areas, a signature of skin alteration which has been suggested to be linked to a defect in MnSOD protective activity. Altogether, those data suggest that the combination of a suitable protective UV filter together with this bioactive Vfe might improve skin protection through complementary pathways.

Thyroid hormone receptor beta1 is expressed in the human hair follicle.

To understand better the mechanisms by which thyroid hormone can exert its effects on the hair follicle, we looked for the expression of members of the thyroid hormone receptor (TR) family in human hair follicles. Immunoreactive TRs were detected in both dermal and epithelial compartments of the human pilosebaceous unit. Using reverse transcriptase-polymerase chain reaction, we established that TRbeta1 was the predominant form of TR expressed in the human hair follicle. In addition, we investigated the effects of 3,3', 5-triiodo-L-thyronine (T3) on the survival of human hair follicles in vitro, to understand the role of this thyroid hormone on hair follicle homeostasis. A physiological level of free T3 significantly enhanced human hair survival in vitro.

Expression of peroxisome proliferator activated receptors (PPARs) in human hair follicles and PPAR alpha involvement in hair growth.

Peroxisome proliferator-activated receptors (PPARs), which belong to the nuclear hormone receptor superfamily, have recently been described as potent key regulators of epidermal development. As 1,25-dihydroxyvitamin D3, retinoic acid and triiodothyronine are known to exert effects on skin and hair follicle growth through similar receptors, we decided to investigate both the expression pattern of the PPAR alpha, -delta and -gamma subtypes and their role in human hair follicles. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry, we established that PPAR alpha, -delta and -gamma were expressed in both dermal and epithelial human hair follicle cells. Additionally, we evaluated the dose effect of clofibrate, a PPAR alpha ligand, on the survival of human hair follicles in culture. A beneficial effect was observed within a narrow range of concentrations.

Expression of retinoid nuclear receptor superfamily members in human hair follicles and its implication in hair growth.

Since clinical evidence of hair loss and hair depigmentation following etretinate therapy has been reported, we decided to study the expression levels of several members of the retinoid nuclear receptor superfamily in dermal and epithelial compartments of the human hair follicle. Additionally, we evaluated the effects of several ligands for these receptors on human hair growth in culture in vitro. We observed that the cellular/ cytoplasmic retinoic acid (RA) binding protein-II and the retinoid-X-receptor-alpha were constantly and strongly expressed in both compartments at levels comparable to those of vitamin D receptor. In dermal papilla cells, by contrast with RAR beta which was always expressed, RAR alpha and RAR gamma were not constantly expressed. In dermal sheath fibroblasts, both RAR alpha, RAR beta and RAR gamma mRNAs were moderately expressed, while in the epithelial compartment, namely the plucked hair, we observed the expression of the same genes in the absence of RAR beta. We also observed that RAR agonists all-trans RA and CD367 inhibited the survival of human hair follicles in culture in vitro, while RXR agonist CD2425 stimulated hair growth and survival at levels comparable to those of 1 alpha,25-dihydroxyvitamin D3, suggesting that RXR agonists might stimulate hair growth in humans in vivo.

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.

Pro-inflammatory cytokine cascade in human plucked hair.

Using reverse transcriptase polymerase chain reaction we showed that freshly plucked human anagen hair expressed both type 1 (80 kD) and type 2 (60 kD) interleukin (IL)-1 receptor mRNAs. The IL-1 receptor type 1 was functional since after in vitro stimulation of plucked hair with IL-1 alpha, we observed the induction of mRNA(s) for the inflammatory cytokines IL-1 beta, tumour necrosis factor alpha and IL-6 as well as for the chemokines monocyte chemotactic and activating factor and IL-8. In addition, the growth of dissected human anagen hairs in culture in vitro was significantly and dose-dependently inhibited by IL-1 alpha as a consequence of hair bulb degradation. These observations, together with those of other authors in IL-1 alpha transgenic mice evidence the inhibitory role of IL-1 on human hair growth. Therefore, in order to identify individuals with high inflammatory potential in their hair follicle environment, we designed a rapid and simple assay to detect variations in the level of IL-1 alpha production in the overnight supernatant of plucked hairs in culture. We observed that 32.7% of the specimens from the volunteers tested (n = 116) could be considered highly inflammatory in terms of IL-1 alpha production. Altogether, these results suggest that in alopecia androgenetica, hair growth might be negatively influenced by IL-1, directly produced by the outer root sheath keratinocytes. Consequently, identifying the "inflammatory alopecic individual' might be of clinical interest to discriminate among individuals for whom anti-IL-1 strategies might be of therapeutic relevance.