Can plant-derived phytochemicals provide symptom relief for hair loss? A critical review.

It is known that hair growth disorders and hair loss can cause personal distress and affect well-being. Whilst clinical conditions remain a target for medical research, current research on hair follicle biology and hair growth control mechanisms also provides opportunities for a range of non-medical and cosmetic interventions that have a modulating effect on the scalp and follicle function. Furthermore, an improvement of the hair fibre characteristics (cuticle structure, cortex size and integrity) could add to the overall positive visual effect of the hair array. Since phytochemicals are a popular choice because of their traditional appeal, this review provides a critical evaluation of the available evidence of their activity for hair benefit, excluding data obtained from animal tests, and offers recommendations on improving study validity and the robustness of data collection in pre-clinical and clinical studies.

La perte des cheveux ou les troubles de la croissance sont connus pour engendrer une grande détresse et affecter le bien-être des individus. Bien que les maladies soient la cible principale de la recherche médicale, de récentes découvertes sur la biologie du follicule pileux et les mécanismes de contrôle de la pousse de cheveux pourraient donner lieu à des interventions non-médicales ou cosmétiques afin d'impacter le cuir chevelu et le follicule. De plus, améliorer les caractéristiques de la fibre capillaire (structure des cuticules, taille et intégrité du cortex) pourrait contribuer à embellir l'apparence de la masse capillaire. Les phytoingrédients sont populaires car traditionnellement utilisés dans les cosmétiques, et cette revue présente une évaluation critique de leurs bénéfices pour les cheveux, en excluant les données issues de tests sur les animaux. Des recommandations afin d'améliorer la collecte de données et la validité des études pré-cliniques et cliniques sont aussi présentées dans cette revue.

Cytokine gene expression in intact anagen rat hair follicles.

Substantial cellular proliferative activity is necessary to produce a mature hair follicle. Therefore, it is likely that cytokines and their receptors play an important controlling role. To provide an understanding of the mechanisms involved during hair growth, we investigated the expression of cytokines in rat anagen hair follicles. A new technique was developed that allowed the rapid isolation of large numbers of intact, viable, anagen, rat pelage hair follicles. Total RNA was isolated from these follicles using an acid-phenol-chloroform extraction and analyzed for cytokine expression. Using the conventional technique of Northern blotting, it was only possible to detect transcripts for transforming growth factor beta (TGF beta) and insulin-like growth factor I (IGFI). Polymerase chain reaction amplification of reverse-transcribed mRNA detected cDNA fragments for TGF beta, IGF I, IGF II, nerve growth factor beta (NGF beta), and interleukin-1 alpha (IL-1 alpha). The amplified products were confirmed by digestion with restriction endonucleases. The proteins themselves for TGF beta and IGF I have been shown to be present within the anagen hair follicle using immunogold antibody labeling. This study has provided the first reported cytokine expression profile of rat anagen hair follicles. It is likely that the analysis of the pattern and timing of expression of these cytokines in the follicle will provide valuable insights into hair growth regulation.

Immune privilege in hair growth.

Immunostaining techniques were used to investigate the relationship between immune cells, proteoglycan, and class I MHC distribution in skin during the hair cycle in rats. The growth stage, anagen, was characterized by absence of class I MHC staining on most cells of the lower follicle and presence of chondroitin proteoglycan in the follicle sheath and dermal papilla. Immune cells were few in number and not associated with follicles. Dramatic changes were observed during regression in catagen; class I MHC was expressed on all follicle epithelium, large numbers of activated macrophages aggregated around the follicles, and the chondroitin proteoglycans disappeared from the follicle sheath and dermal papilla. During the resting stage, telogen, class I MHC remained on cells of the secondary germ, but macrophages and chondroitin proteoglycans were absent. These observations lead us to propose a hypothesis of immune privilege in hair growth.

Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes.

Autoantibodies to a normal component of stratified squamous epithelia, the bullous pemphigoid antigen (BPA), are synthesized in patients with the disease bullous pemphigoid. We have used these sera to study the distribution of BPA in vivo and in vitro. At low magnification, indirect immunofluorescent staining for BPA is linear at the basement membrane zone (BMZ) of skin and many other epithelial tissues. At higher magnification however, we observed a punctate staining pattern for BPA which was regular in appearance and suggested localization of BPA to discrete structures at the BMZ. Subsequent immunoelectron microscopy using both peroxidase and colloidal gold labeling techniques with patients' sera or IgG, revealed that BPA is associated with hemidesmosomes--putative adhesion structures at the BMZ, based on their similarity in ultrastructure to desmosomes. More specifically BPA was immunolocalized to the cytoplasmic face of hemidesmosomes and was not observed extracellularly in the basement membrane. In stratifying and nonstratifying cultures of rat keratinocytes, BPA is expressed intracellularly and not in the cell-derived matrix, unlike other known basement membrane components. These cells also synthesize BPA in vitro, and immunoprecipitation from metabolically labeled cultures revealed a 220 kD polypeptide under reducing conditions. From these observations we conclude (1) that BPA is a 220 kD polypeptide component either of or associated with hemidesmosomes, and (2) that it is localized intracellularly both in vivo and in vitro. We suggest that BPA is not normally a lamina lucida component, but that it may form part of a linkage between the cytoskeleton and the basement membrane.

Immunohistochemical localization of basement membrane components during hair follicle morphogenesis.

Specific antisera were used to investigate the distributions of several basement membrane zone (BMZ) components, namely, bullous pemphigoid antigen (BPA), heparan sulfate proteoglycan (HSPG), laminin, and type IV collagen, during the development of hair follicles in late embryo rats. BPA was not visible by indirect immunofluorescence in the BMZ before epidermal involution but appeared in all regions of BMZ after this had occurred. As follicular length increased during maturation, the distribution of BPA was no longer uniform, being reduced or absent from the BMZ around the lower part of the elongating follicle. HSPG was associated with the basal cell layer prior to the appearance of hair follicle primordia and became BMZ-associated before birth but after follicle buds were first observed. HSPG was also found to be associated with the basal cell surfaces in the epidermis, but not in the hair follicle. Laminin and type IV collagen were continually present in epidermal and follicular BMZ both before and during development of hair follicles and were later present in the dermal papilla matrix. From these observations we conclude that (1) laminin and type IV collagen are functionally important for BMZ integrity before and during epidermal and follicular maturation, (2) HSPG may have a cell surface function in epidermis as well as roles in BMZ organization and properties, and (3) the distribution of BPA is indicative of its association only with regions of tissue not involved in morphogenetic change. We also suggest that the cell-matrix interactions documented for BPA, HSPG, laminin, and fibronectin may depend on the type of tissue involved and its state of development, differentiation, or repair.

In the absence of streptomycin, minoxidil potentiates the mitogenic effects of fetal calf serum, insulin-like growth factor 1, and platelet-derived growth factor on NIH 3T3 fibroblasts in a K+ channel-dependent fashion.

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

Distribution of proteoglycans during the hair growth cycle in human skin.

The involvement of proteoglycans in hair growth has been recognized through the observation of increased hair growth in diseases such as the mucopolysaccharidoses and pre-tibial myxedema, which involve an increase in skin proteoglycan content. In an attempt to understand this, we have examined the distribution of chondroitin 6 sulphate (C6S), unsulphated chondroitin (COS), dermatan sulphate (DS), and heparan sulphate proteoglycans (HSPG) in frozen tissue sections of normal scalp by immunostaining. Results show that during anagen, the thick connective tissue sheath around the follicle strains strongly for C6S, COS, and DS. COS is uniquely associated with this region and is not found beneath the epidermis or infundibular epithelium. HSPG is, however, localized in the basement membrane zone adjacent to the outer root sheath. In addition, all of these proteoglycans are localized in the dermal papilla. In mid-catagen, we observed significant loss of C6S and COS staining from both the dermal papilla and the connective tissue sheath, but no decrease in staining for HSPG. In late catagen, very little staining of C6S and COS was observed. In early anagen, we observed that C6S was again present in the connective tissue sheath and dermal papilla; however, COS staining appeared to be weaker and less closely associated with the follicle. HSPG staining was observed in early anagen in a pattern very similar to that found for other basement membrane components. Results for DS were not obtained for catagen or early anagen. These results provide further evidence that hair growth is associated with the presence of chondroitin proteoglycans in the follicle environment and that the cessation of growth is associated with their removal. Further studies are underway to characterize the relationship between hair growth and proteoglycans.

Rapid isolation in large numbers of intact, viable, individual hair follicles from skin: biochemical and ultrastructural characterization.

A rapid, novel method is described by which large numbers of intact, viable, individual hair follicles may be isolated from rat skin. Follicles are freed from the surrounding connective tissue by shearing, which is effected by repeated cutting with a loosely fitting pair of scissors, and collected individually under liquid using gentle aspiration. Ultrastructural analysis indicates that the follicles are sheared away from the surrounding dermis in the region of the connective tissue capsule which encircles the hair. The follicles appear viable by light and electron microscopy and, within 2 h of isolation, retain the capacity to incorporate [3H]thymidine into DNA and [35S]methionine into proteins as judged by autoradiography. A histologic comparison indicates that the structural integrity of follicles isolated by this new method is significantly superior to those plucked from the animal at the same time. The method affords the isolation of large numbers of hair follicles, without resort to enzyme treatments, suitable for biologic studies in the absence of other skin appendages and dermis.

Human hair growth in vitro: a model for the study of hair follicle biology.

The factors that regulate hair follicle growth are still poorly understood. In vitro models may be useful in elucidating some aspects of hair follicle biology. We have developed an in vitro human hair growth model that enables us to maintain isolated human hair follicles for up to 10 days, during which time they continue to grow at an in vivo rate producing a keratinised hair fibre. We have shown that epidermal growth factor (EGF) in our system mimics the in vivo depilatory action of EGF in sheep, and suggest that this occurs as a result of EGF stimulating outer root sheath (ORS) cell proliferation which results in the disruption of normal mechanisms of cell-cell interaction in the hair follicle. We identify transforming growth factor-beta (TGF-beta) as a possible negative regulator of hair follicle growth and show that physiological levels of insulin-like growth factor-I (IGF-I) can support the same rates of hair follicle growth as supraphysiological levels of insulin. Furthermore, in the absence of insulin hair follicles show premature entry into a catagen-like state. This is prevented by physiological levels of IGF-I. Finally we demonstrate that the hair follicle is an aerobic glycolytic, glutaminolytic tissue and discuss the possible implications of this metabolism.

An in vitro model for the study of human hair growth.

Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermosubcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watch makers' forceps. Isolated hair follicles maintained free floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinized hair shaft, and was not associated with the loss of hair follicle morphology. [Methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labeling of keratins showed that their patterns of synthesis did not change with maintenance. Serum was found to inhibit hair follicle growth in vitro; and when follicles were maintained in serum-free medium, they grew for up to 10 days, suggesting that in vitro the hair follicles are able to regulate their own growth, possibly by the production of relevant growth factors. This may prove useful in identifying the autocrine/paracrine mechanisms that operate in the hair follicle. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth regulatory effect on hair follicles in vitro and that EGF and its other receptor ligand TGF-alpha mimic the in vivo depilatory effects of EGF that have been reported for sheep and mice.

Glycosaminoglycan synthesis by cultured human hair follicle dermal papilla cells: comparison with non-follicular dermal fibroblasts.

The extracellular matrix of the hair follicle dermal papilla is rich in glycosaminoglycans, the expression of which varies during the hair growth cycle being maximal in anagen and becoming undetectable as the follicle enters telogen. These observations, together with other experimental and clinical evidence, suggest that glycosaminoglycans may be involved in regulating hair growth. To investigate the metabolism of glycosaminoglycans by the dermal papilla we have measured the incorporation of radiolabelled precursors into glycosaminoglycans released into extracellular matrix and culture medium by cultured human dermal papilla cells. We also studied glycosaminoglycan synthesis by cells cultured from the lower follicular connective tissue sheath and by non-follicular dermal fibroblasts. Compared with dermal fibroblasts, dermal papilla cells showed a three to fourfold higher level of incorporation of 35S-sulphate and 3H-glucosamine into extracellular matrix glycosaminoglycans. Dermal papilla cells also released more 3H-glucosamine-labelled glycosaminoglycan into culture medium than dermal fibroblasts but there was no difference in 35S-sulphate labelling. These findings indicate that dermal papilla cells maintain a high level of glycosaminoglycan synthesis in vitro. Specific enzyme/chemical degradation showed that dermal papilla cells and dermal fibroblasts synthesized the same glycosaminoglycan types. However, the results suggested that dermal papilla glycosaminoglycans are less sulphated than those synthesized by dermal fibroblasts and that a higher proportion of sulphated glycosaminoglycans is retained in an extracellular matrix. The synthesis of glycosaminoglycans by connective tissue sheath cells was similar to that of dermal papilla cells, supporting the view that the dermal papilla and connective tissue sheath share certain properties.

Prolonged maintenance of human hair follicles in vitro in a serum-free medium.

We have previously reported the in vitro growth of human hair follicles for up to 4 days in a partially defined medium containing serum. We now report the prolonged in vitro growth of isolated human hair follicles for at least 9 days. This was achieved after analysis of the contribution of certain components of the original medium and, by a process of elimination, deriving a completely defined medium supplemented only with antibiotics, L-glutamine, insulin and hydrocortisone. We have shown, by [methyl-3H] thymidine autoradiography, that the hair follicles grown in this medium maintain an in vivo pattern of DNA synthesis, and that the gross morphology and histology of these maintained hair follicles remains similar to that of freshly isolated hair follicles. We have also shown that the patterns of keratin synthesis, as determined by [35S] methionine labelling, do not alter with maintenance.

Characterization of LHTric-1, a new monospecific monoclonal antibody to the trichocyte keratin Ha1.

The hair follicle is a heterogeneous tissue involving differentiation of both hair forming (trichocyte) and non-hair forming (root sheath) cells; while there are many antibody markers available which can determine the distribution of 'soft' epithelial keratins, fewer have been described which are truly monospecific for hair specific 'hard' keratins. We employed the proven strategy of raising monoclonal antibodies to a short synthetic peptide from the carboxy-terminal sequence of mouse Ha1 and report here the successful production of a monospecific monoclonal antibody which we have called LHTric-1. We have characterized the antibody using immunostaining on rat and human tissues and by immunoblotting against an extract of human follicles. The antibody cross-reacted between rat and human tissue but did not stain formalin-fixed tissue. LHTric-1 localized very specifically to the pre-cortical region of the hair follicle in early anagen and to pre-cortical cells in the upper bulb in anagen. Telogen follicles did not react. LHTric-1 immunoreacted within tongue and nail, staining being restricted to the mid-line above the connective tissue core in tongue and to the suprabasal layers of the nail matrix. The antibody did not react with the fully keratinized hair or nail plate. Finally, in immunoblotting, LHTric-1 reacted with a single band of 44 kDa, suggesting that a single protein was recognized. We conclude that this antibody, by virtue of its known antigen sequence specificity, will be useful in research into the formation of hair and nail in normal and diseased states.

Keratin expression in the normal nail unit: markers of regional differentiation.

Differentiation within the nail unit was examined using a range of antikeratin monoclonal antibodies including the recently described antibody LHTric-1, specific to the acidic hair-type keratin Ha1. Keratinocytes of the nail matrix, nail bed and the digit pulp were characterized by different patterns of keratin expression. Nail matrix was the sole site of expression of Ha1, which colocalized in suprabasal matrix epidermis with epidermal keratins K1 and K10. Small amounts of K17 were found at the apex of the matrix in some cases. K6 and K16 were found where the epidermal surface folds forwards to become the ventral aspect of the proximal nail fold. The nail bed was distinguished by the absence of hair-type keratin Ha1 and the absence of markers of cornified epidermis and mucosal differentiation K1/K10 and K4/K13, respectively, while K6, K16 and K17 were detected. The basal keratin conformation marker, LH6, was expressed suprabasally throughout the nail bed. This complement of keratins exists in the nail bed in the absence of notable proliferative activity, and suggests a state of minimally developed differentiation which may be afforded by the physical or biological properties of the overlying nail. Keratins, K6, K16 and K17 were all found in the digit pulp in limited amounts, possibly in association with the epidermal component of the eccrine duct. The simple epithelial keratins, K7, K8 and K18, were found in small amounts in the specimens from younger individuals, mainly in epibasal cells of the apex of the matrix and in putative Merkel cells.

Extracellular matrix derived from hair and skin fibroblasts stimulates human skin melanocyte tyrosinase activity.

There is indirect evidence that both skin and hair melanocytes are regulated by the activity of adjacent cells. In hair, the specialized fibroblasts (dermal papilla cells) appear to play a role in the regulation of hair growth. Hair pigmentation may relate to hair growth. In skin, melanocytes are located adjacent to the basement membrane zone. As far as we are aware, direct interactions of fibroblasts with melanocytes have not previously been investigated. Accordingly, the objective of this study was to develop co-culture conditions in which to investigate whether dermal fibroblasts from skin or hair could influence melanocyte differentiation. The influence of fibroblast-conditioned media, co-culture with fibroblasts, and fibroblast-derived extracellular matrix (ECM) on normal human skin melanocyte tyrosinase activity was examined. Fibroblasts from both skin and hair were capable of altering melanocyte morphology and significantly increasing tyrosinase activity when melanocytes were cultured in the absence, but not the presence, of the major proliferative drives. Although stimulation of tyrosinase activity was detectable with conditioned medium and co-culture with fibroblasts, the most striking result was obtained with the fibroblast-produced ECM which, on average, produced a four-fold increase in tyrosinase activity within 6 days. Thus, the study describes co-culture conditions in which the stimulatory effect of the fibroblast on melanocyte differentiation can be examined.