A prostaglandin D-synthase-positive mast cell gradient characterizes scalp patterning.

BACKGROUND: Pattern (androgenetic) alopecia is commonly encountered in scalp biopsies obtained for non-scarring hair loss. Prostaglandin D-synthase is known to be elevated in bald vs. non-alopetic scalp of patients with androgenetic alopecia. We hypothesized that this difference in pattern of prostaglandin D-synthase expression may constitute a developmental pattern inherent to normal as well as alopecic scalp skin, thus defining a 'field' vulnerable to acquired hair loss. METHODS: We immunohistochemically mapped prostaglandin D-synthase expression from supra-auricular to vertex scalp skin of 11 cadavers. RESULTS: We found significantly more dermal mast cells immunoreactive for prostaglandin D-synthase in the vertex compared to the lateral aspects of the scalp, with a decrement that spatially approximated the pattern of androgenetic alopecia. This difference was present in both balding and non-balding scalps and was independent of gender. Dual labeling established dermal cells expressing prostaglandin D-synthase as mast cells. CONCLUSIONS: These data indicate that scalp is spatially programmed via mast cell prostaglandin D-synthase distribution in a manner reminiscent of the pattern seen in androgenetic alopecia.

Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia.

Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D(2) synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D(2) (PGD(2)), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD(2) levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD(2) inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD(2) receptor G protein (heterotrimeric guanine nucleotide)-coupled receptor 44 (GPR44), but not the PGD(2) receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD(2) in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD(2) as an inhibitor of hair growth in AGA and suggest the PGD(2)-GPR44 pathway as a potential target for treatment.

Different patterns of 5alpha-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells.

Androgens regulate hair growth, and 5alpha-reductase (5alphaR) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5alphaR activity. Real-time RT-PCR revealed that the highest level of 5alphaR1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5alphaR1 mRNA was observed in fibroblasts. Minimally detectable levels of 5alphaR2 mRNA were found in all three cell types. A weak band at 26kDa corresponding to the human 5alphaR1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5alphaR1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5alphaR assay using [(14)C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5alphaR activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17beta-HSD rather than 5alphaR among the three cell types. The 5alphaR1 inhibitor MK386 exhibited a more potent inhibitory effect on 5alphaR activity than finasteride (5alphaR2 inhibitor) in bDPCs.

Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions.

Organ culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William's E medium for more than 2 weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM) + 10% fetal bovine serum (FBS). Moreover, William's E medium protected better against cell death than MEM + 10% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13 days in culture. The percentage of surviving hair follicles (mostly with catagen- or telogen-like morphology) gradually increased over time displaying mostly catagen hair follicles after 17 days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long-term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2 weeks under well-defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.

Human scalp dermal papilla and fibrous sheath cells have a different expression profile of matrix metalloproteinases in vitro when compared to scalp dermal fibroblasts.

The dermal papilla is a cluster of specialised mesenchymal cells at the bottom of the mammalian hair follicle, embedded in a loose extracellular matrix. These cells have the capability to induce and support hair growth via close epithelial-mesenchymal interactions with the keratinocytes surrounding the hair matrix. The extracellular matrix of the dermal papilla differs markedly from the interfollicular matrix and plays a key role in the maintenance of hair growth. In this study we investigated the expression pattern and activity of matrix metalloproteinases (MMP) and their tissue inhibitor in in vitro cultures of cells derived from scalp dermal papilla and fibrous sheath. Expression and activity of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2 and MT1-MMP were analysed in those cells cultured in contact with one of the relevant protein component of the dermal matrix, collagen type I as well as in monolayer. Zymographic analysis showed activation of MMP-2 in all cells grown in three-dimensional collagen lattices whereas MMP-9 was activated only in three-dimensional collagen cultures of dermal fibroblasts and weakly in follicular cells. Expression of MMP-1, TIMP-1, TIMP-2 and MT1-MMP was similar in all cells, in both culture conditions, whereas expression of MMP-3 was absent in dermal papilla cells. In addition to a series of reported morphological and functional differences between dermal fibroblasts and the dermal mesenchyme-derived cells of the hair follicle, we reported differences in MMP expression in dermal papilla and fibrous sheath cells within the mesenchymal population of the hair follicle.

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis.

BACKGROUND: Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. OBJECTIVES: We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. METHODS: This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). RESULTS: Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. CONCLUSIONS: The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.

The 5alpha-reductase type 1, but not type 2, gene is expressed in anagen hairs plucked from the vertex area of the scalp of hirsute women and normal individuals.

The aim of the present study was to determine the expression of the genes for type 1 (SDR5A1) and type 2 (SDR5A2) 5alpha-reductase isoenzymes in scalp hairs plucked from 33 hirsute patients (20 with polycystic ovary syndrome and 13 with idiopathic hirsutism) and compare it with that of 10 men and 15 normal women. SDR5A1 and SDR5A2 expression was estimated by RT-PCR using the gene of the ubiquitously expressed protein 2-microglobulin as an internal control. The results are expressed as arbitrary units in relation to beta2-microglobulin absorbance (mean SEM). SDR5A2 expression was not detected in any hair samples analyzed in this study. No differences were found in SDR5A1 mRNA levels between men and normal women (0.78+/-0.05 vs 0.74+/-0.06, respectively). SDR5A1 gene expression in the cells of hair plucked from the scalp of normal women (0.85+/-0.04) and of women with polycystic ovary syndrome (0.78+/-0.05) and idiopathic hirsutism (0.80+/-0.06) was also similar. These results indicate that SDR5A1 gene expression in the follicular keratinocytes from the vertex area of the scalp seems not to be related to the differences in hair growth observed between normal men and women and hirsute patients. Further studies are needed to investigate the expression of the 5alpha-reductase genes in other scalp follicular compartments such as dermal papillae, and also in hair follicles from other body sites, in order to elucidate the mechanism of androgen action on the hair growth process and related diseases.

5 alpha-reductase type 2 is constitutively expressed in the dermal papilla and connective tissue sheath of the hair follicle in vivo but not during culture in vitro.

Recent studies suggest that 5 alpha-reductase type 2 (5 alpha R2) rather than 5 alpha R1 plays a key role in the pathogenesis of male-pattern baldness. To clarify the localization of the androgen receptor (AR), 5 alpha R1, and 5 alpha R2 in the hair follicle, we investigated the expression of the corresponding genes by RT-PCR using microdissected hair follicles. AR and 5 alpha R1 mRNAs were expressed in all portions of the hair follicle. By contrast, 5 alpha R2 mRNA was expressed only in mesenchymal portions that included the dermal papilla and connective tissue sheath, and hardly any was expressed in epithelial portions. The intensity of expression of these genes in each portion of the hair follicles did not differ between follicles from balding and nonbalding scalp. We also examined the expression of these genes in cultured fibroblasts derived from the dermal papilla and connective tissue sheath. Although expression of AR and 5 alpha R1 mRNAs was easily detected, there was no obvious expression of 5 alpha R2 mRNA in either type of cell. Type-specific inhibition of 5 alpha R activity by MK386 and MK906 confirmed these patterns of expression of 5 alpha R mRNA. Thus, the expression of 5 alpha R2 mRNA seems to be characteristic of freshly microdissected mesenchymal portions of the hair follicle, but such expression might not be maintained in culture.

Clinical application of 5alpha-reductase inhibitors.

The inhibitors of 5alpha-reductase isoenzymes (1 and 2) can be schematically divided in three groups according they substrate specificity: a) pure or preferential inhibitor of 5alpha-reductase 1; b) pure or preferential inhibitor of 5alpha-reductase 2; c) dual inhibitors. Despite the fact that several steroidal and non-steroidal inhibitors have been synthesized and experimented in pharmacological models, only finasteride has been extensively used for clinical purposes. The largest application of finasteride in man has been human benign prostative hyperplasia (BPH). In addition, finasteride has been recently used for treatment of male baldness with a 50% of objective response. In women, finasteride has been used in some control trials for treatment of hirsutism with an objective favorable response. In conclusion, finasteride appears be useful for BPH, baldness and hirsutism (with caution) treatment. On the basis of experimental observations on distribution of 1 and 2 isoenzymes in human skin, scalp and prostate, the dual inhibitors should be more indicated for treatment of BPH and baldness. Similarly, the dual inhibitors seem indicated in attempting to prevent prostatic cancer. The pure 5alpha-reductase 1 inhibitors seem the ideal drugs for treatment of acne and hirsutism.

Expression of neuropeptide-degrading enzymes in alopecia areata: an immunohistochemical study.

BACKGROUND: Much clinical evidence suggests that the nervous system, including psychological factors, can influence the course of alopecia areata (AA). However, there has been little substantial evidence of specific participation of cutaneous neurogenic factors in the disease process. OBJECTIVES: As previous studies have demonstrated that stress elicits the release of the neuropeptide substance P (SP) from peripheral nerves and that some patients with AA show prominent SP expression in nerves surrounding their hair follicles, we aimed to evaluate the role of SP in AA. METHODS: We used immunohistochemistry to examine the expression of SP and SP-degrading enzymes in scalp biopsies from patients with AA and from healthy controls. RESULTS: Affected hair follicles in the centre of the areas of hair loss of patients with AA were richly innervated by SP-staining nerve fibres. Strong expression of the SP-degrading enzyme, neutral endopeptidase (NEP), was observed in hair follicles not only in the acute progressive phase of AA but also in the chronic stable phase. Expression of NEP in hair follicles from the margins of areas of hair loss was stronger than in normal controls, but was weaker than in the centre of the areas of hair loss. In addition, endothelial immunoreactivity for angiotensin-converting enzyme (also capable of degrading SP) was not observed in the centre of the areas of hair loss, which was in significant contrast to normal controls as well as to the margins of areas of hair loss where it was expressed. Further, intense expression of endothelial leucocyte adhesion molecule-1 on vessels and many degranulating mast cells was observed adjacent to affected hair follicles in AA, in admixture with dense lymphocytic inflammation. CONCLUSIONS: These findings suggest that SP is endogenously released by dermal nerve fibres around hair follicles and that it may play an important part in epithelial-mesenchymal-neuroectodermal interactions in AA. This study reveals that SP and its degrading enzymes are involved in the pathogenesis of AA, which in turn might explain the pathological significance of neurogenic and psychogenic aspects in the disease process.

Differentiation of hair growth cycle from scalp hair roots for the diagnosis of glucose-6-phosphate dehydrogenase deficiency in neonates.

Hair analysis can be used as a screening tool in the diagnosis of genetic diseases. The scalp hair roots of 67 normal neonates and 39 neonates with glucose-6-phosphate dehydrogenase (G6PD) deficiency were analysed using Fourier transform infrared (FT-IR) microspectroscopy to differentiate the stages of the hair growth cycle and to diagnose the genetic disorder on the basis of spectral differences. We have demonstrated that FT-IR microspectroscopy is a rapid and effective noninvasive diagnostic method to differentiate scalp hair roots of normal neonates into the anagen, catagen or telogen phases of the hair growth cycle, using IR spectral differences within the 3000-2800 cm(-1) region and the IR peak area ratio of 2854 cm(-1)/2873 cm(-1) or 1084 cm(-1)/amide II band (p<0.001). Moreover, G6PD-deficient neonates could be accurately diagnosed from telogen phase hair roots owing to significant differences in IR peak area ratios of 2854 cm(-1)/2873(-1) or 1084 cm(-1)/amide II band compared to normal values in healthy neonates. The result suggests that the application of FT-IR microspectroscopy may be capable not only of differentiating the hair growth cycle into anagen, categen or telogen phases but also of detecting G6PD deficiency. Hair root analysis promises to be a useful complement to serum and urine analysis in the diagnosis of genetic diseases.

Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5alpha-reductase.

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.

Identification of tyrosine hydroxylase as an autoantigen in autoimmune polyendocrine syndrome type I.

Patients with the autosomal recessively inherited autoimmune polyendocrine syndrome type I (APS I) have autoantibodies directed against several endocrine and nonendocrine organs. In this study a new autoantigen related to this syndrome, tyrosine hydroxylase, was identified in sera from patients with alopecia areata through immunoscreening of a scalp cDNA library. Immunoreactivity against in vitro expressed tyrosine hydroxylase was found in 41 (44%) of the 94 APS I patients studied and this reactivity correlated with the presence of alopecia areata (P = 0.02). These findings further stress the importance of enzymes involved in neurotransmitter biosynthesis as important immune targets in APS I.

Immunohistochemical localization of types 1 and 2 5alpha-reductase in human scalp.

The predominant form of 5alpha-reductase (5aR) in human scalp is 5aR1. None the less, clinical studies have shown that finasteride, a selective inhibitor of 5aR2, decreases scalp dihydrotestosterone and promotes hair growth in men with androgenetic alopecia. Immunolocalization studies were thus carried out to examine 5aR isozyme distribution within scalp and, in particular, to determine whether 5aR2 might be associated with hair follicles. 5aR2 was localized using both a rabbit polyclonal and a mouse monoclonal antibody. 5aR1 was detected with a mouse monoclonal antibody. The specificity of these reagents was demonstrated both by immunofluorescence and Western blot analyses of COS cells overexpressing human 5aR1 or 5aR2. When cryosections of scalp from men with androgenetic alopecia were stained with antibody against 5aR2, using immunoperoxidase avidin-biotin complex methodology, immunostaining was observed in the inner layer of the outer root sheath and, in more proximal regions of the follicle, in the inner root sheath. Staining was also prominent in the infundibular region of the follicle, with less intense staining extending throughout the granular layer of the epidermis. Some staining was also seen in sebaceous ducts. Similar results were obtained with both the polyclonal and monoclonal 5aR2 antibodies. In contrast, in scalp cryosections stained with antibody to 5aR1, no immunostaining was observed within hair follicles. Intense staining for the type 1 isozyme was, however, detected within sebaceous glands. Our immunolocalization data suggest that the results seen in clinical trials of men with male pattern hair loss treated with finasteride may be due, at least in part, to local inhibition of 5aR2 within the hair follicle.

Minoxidil increases 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase activity of cultured human dermal papilla cells from balding scalp.

Minoxidil is known to induce hair growth in male pattern baldness, for which development androgen plays a central role. We studied the effect of minoxidil on testosterone metabolism by cultured dermal papilla cells from balding or nonbalding scalp and dermal fibroblasts. In all three groups, 17beta-hydroxysteroid dehydrogenase activity was much higher than 5alpha-reductase activity. Minoxidil increased 17beta-hydroxysteroid dehydrogenase activity by nearly 40% (P < 0.001) in dermal papilla cells of balding scalp, whereas the effect was less marked in dermal papilla cells from nonbalding scalp and dermal fibroblasts. 5alpha-Reductase activity was also slightly increased by minoxidil in dermal papilla cells from balding scalp. Again, the effect on 5alpha-reductase activity was insignificant in the other two groups of cells. Whether such modification of testosterone metabolism in dermal papilla cells of balding scalp by minoxidil is related to its therapeutic effect remains unknown.

Expression of transglutaminase 1 in human hair follicles, sebaceous glands and sweat glands.

To explore the function of the enzyme transglutaminase 1 (TGase 1), its distribution was analysed by immunofluorescence microscopy, postembedding immunoelectron microscopy and in situ hybridization. TGase 1 was expressed in the outer root sheath (ORS) cells in the distal portion of the isthmus and infundibulum of human hair follicles. In the level of the proximal and middle portion of the isthmus, TGase 1 was observed in the keratinized area of the inner root sheath (IRS) and ORS cells. In the bulbar and suprabulbar portions, TGase 1 was present in the ORS and IRS cells. The cortex and medulla cells in these regions also contained TGase 1. A high level of fluorescence was observed at the cuticle of the cortex. Sebaceous and sweat gland cells contained abundant TGase 1. Possible functions of TGase 1 in these epidermal appendages are discussed.

Synthesis and 5 alpha-reductase inhibitory activity of 8-substituted benzo[f]quinolinones derived from palladium mediated coupling reactions.

Benzoquinolinones have been shown to be potent, selective inhibitors of the Type I 5 alpha-reductase enzyme, which is responsible for the production of dihydrotestosterone from testosterone localized in the scalp. In an effort to identify compounds that demonstrate inhibition of both 5 alpha-reductase isozymes, we have employed 8-bromobenzoquinolinone as an advanced intermediate for participation in a variety of palladium mediated carbon-carbon bond forming reactions. By varying the 8-substituent it is possible to alter the selectivity profile of the series.

LY191704 inhibits type I steroid 5 alpha-reductase in human scalp.

Conversion of testosterone to dihydrotestosterone (DHT) has been demonstrated to be catalyzed by two isoforms of steroid 5 alpha-reductase, designated types I and II. Although several classes of steroid-based inhibitors of the type II isoform have been identified, these agents have not demonstrated highly selective pharmacological activity against human type I 5 alpha-reductase. LY191704 is representative of a series of nonsteroidal agents that have potent [apparent inhibitory constant (Ki) = 11.3 nM] inhibitory activity in human scalp skin homogenates (pH 7.5), a source of type I 5 alpha-reductase. [3H]-DHT production in the presence and absence of LY191704 is consistent with a noncompetitive mode of inhibition. In human prostatic homogenates (pH 5.5), a source of type II 5 alpha-reductase, LY191704 is virtually inactive as an inhibitor [concentration of inhibitor producing 50% inhibition of enzymatic activity (IC50) > 1,000 nM] of [3H]-DHT formation. LY191704 does not inhibit the type I or type II isoforms of rat 5 alpha-reductase, nor does the compound compete for binding to the murine androgen receptor expressed in SF9 cells using a baculo virus expression system. The benzoquinolinones, as exemplified by LY191704, possess exquisite pharmacological selectivity and provide a tool to understand the role of human type I 5 alpha-reductase in normal and pathophysiological states. These agents may also find clinical utility in treating androgen-dependent dermatological conditions.