A NEW STRATEGY TO MODULATE ALOPECIA 53 repertoire of functions. Their interaction with integrin receptors at the surface of cells anchors skin cells to basement membranes and modulates cellular processes such as pro- liferation, apoptosis, differentiation, and motility (35). Laminin-511 (laminin-10) is the predominant laminin at the epithelial–mesenchymal interface of the hair follicle and is crucial for hair morphogenesis and anagen hair growth (36). Laminin-511 stimulates hair growth in vitro, and preventing its expression at the dermal papilla leads to hair regres- sion and alopecia, a defect that can be prevented by application of the purifi ed protein (37). Chemotherapy-induced alopecia has recently been associated with downregulation of laminin-511 expression, providing a new target for the prevention of this adverse effect (37). The effects of the matrix protein on hair follicle are mediated through the binding and activation of specifi c integrins at the surface of cells (38). Collagen VII. Collagen VII is a long-chain collagen synthesized and secreted by keratino- cytes and dermal fi broblasts. This matrix protein is a major component of anchoring fi brils that are specialized attachment complexes at epithelium–mesenchyme interface, in a number of tissues. In human skin, anchoring fi brils extend from the lower portion of the dermal–epidermal basement membrane to the underlying upper papillary dermis, form- ing U-like structures that entrap interstitial collagen fi bers (39). In the hair follicle, col- lagen VII is found along the BMZ outside the hair follicle and at the dermal papilla junction inside the hair bulb. Collagen VII colocalized with integrins and laminins-511 at the epithelial–mes enchymal interface, suggesting a role in hair growth (14,15). The close interaction of collagen VII with laminins and interstitial collagen provides stability to the epithelial–mesenchymal interface. Effects of acetyl tetrapeptide-3 on matrix proteins. The effect of acetyl tetrapeptide-3 (10−7 M) on the protein expression of type III collagen and total laminins in human fi broblasts (MRC5) was evaluated by selective immunofl uorescence in comparison with untreated fi broblasts. Results showed a signifi cant stimulation in the synthesis of both ECM pro- teins in the presence of the peptide (Figure 4) expression of type III collagen was in- creased by +65% while expression of laminins raised by +285% over untreated cells. Figure 4. Effects of acetyl tetrapeptide-3 (10−7 M) on the protein expression levels of type III collagen and total laminins in human fi broblasts (MRC5) (A) immunofl uorescence staining of type III collagen and lami- nins in control and acetyl tetrapeptide-3-treated cells (B) histogram representation of results. Untreated cells served as control.

JOURNAL OF COSMETIC SCIENCE 54 The potential effect of acetyl tetrapeptide-3 on protein expression levels of collagen VII at a mesenchymal–epithelial junction was documented using human skin explants. Skin atrophy was fi rst induced through corticoid application in an effort to mimic the atrophic state of a defi cient hair follicle. The explants were then treated with acetyl tetrapeptide-3, and collagen VII expression at the DEJ was revealed semiquantitatively by immunohistological staining. Microscopic observations of normal untreated skin revealed a strong labeling of collagen VII along the DEJ. Corticoid treatment resulted in a drastic and signifi cant diminution (−70% on visual scoring) of collagen VII stain- ing but baseline expression was completely restored in the presence of acetyl tetrapep- tide-3 (Figure 5). Given the importance of collagen III, collagen VII, and laminins for hair morphogenesis and growth, the positive impact that acetyl tetrapeptide-3 had on their expression in vitro and ex vivo supports the use of the peptide to help maintain an adequate ECM bed for optimized hair anchorage. INHIBITION OF INFLAMMATORY SIGNALS As mentioned in the Introduction section, microinfl ammation in the vicinity of the der- mal papilla appears to precipitate or at least contribute to male pattern alopecia. To mimic infl ammatory events that may prevail in this context, NHDFs were stimulated with IL-1α in the presence or absence of red clover extract alone, or a mixture of red clover extract and acetyl tetrapeptide-3, at two different concentrations (0.5% and 1%). DMS, a glucocorticoid with anti-infl ammatory properties, served as positive control for inhibition of IL-8 secretion. As was expected, within 24 h of IL-1α stimulation of NHDF cells, a massive release of IL-8 was observed in the culture media, an effect that was partially blocked by DMS (−17%). The production of IL-8 was also slightly inhibited in the presence of increasing concentrations (0.5% and 1%) of red clover extract alone (−11% and −20%, respec- tively). However, the mixture of red clover extract and acetyl tetrapeptide-3 was much more potent and showed a dose–effect relationship, inhibiting IL-8 release by −33% and −48%, at the lowest and the highest concentrations, respectively (Figure 6). From this Figure 5. Effects of acetyl tetrapeptide-3 on protein expression levels of collagen VII at the DEJ, as mea- sured by immunohistological labeling of human skin explants: (A) normal skin (B) corticoid-treated (0.05%) skin (C) corticoid-treated (0.05%) skin, cultured in the presence of acetyl tetrapeptide-3 and (D) visual scoring of collagen VII expression.