Human Scalp Skin Research Articles

P24 Folliculin silencing in human hair follicles ex vivo upregulates intrafollicular mechanistic target of rapamycin complex 1 activity: a new model for studying Birt–Hogg–Dubé syndrome

Abstract Introduction and aims Birt–Hogg–Dubé (BHD) syndrome is a rare multisystem disorder caused by germline pathogenic variants in the FLCN gene. BHD skin tumours predominantly affect facial skin, confer a significant psychosocial burden, and there is a lack of nonsurgical therapies. Here we report a novel ex vivo human model to study the impact of FLCN loss in human hair follicles (HFs), the putative origin of BHD skin tumours. Methods Organ-cultured human anagen HFs and scalp skin were treated with small interfering RNA targeting FLCN (n = 2 donors) for 6 days ex vivo, followed by quantitative real-time polymerase chain reaction (qRT-PCR), and quantitative immunohistomorphometry (qIHM). Results Successful FLCN knockdown was confirmed at both the mRNA (34% reduction compared with nontargeting control levels measured by qRT-PCR) and protein level (12% reduction in FLCN expression measured by qIHM). In FLCN-silenced HFs (n = 14, two donors), our preliminary Results demonstrated an increase in p-S6 expression, a direct downstream kinase of mechanistic target of rapamycin complex (mTORC)1 signalling. This was spatially restricted to the hair bulb and proximal outer root sheath keratinocytes, and was significantly greater than quantified in nontargeting control HFs (P < 0.05). Conclusions FLCN silencing in ex vivo cultured, healthy human HFs and scalp skin provides a novel, instructive and accessible preclinical research model. We aim to delineate whether FLCN interacts with the key endogenous inhibitor of mTORC1 activity, TSC2, in future work, to gain insight into the role of FLCN in the pathobiology of BHD and the maintenance of healthy human HFs.

Interleukin-15 is a hair follicle immune privilege guardian

The autoimmunity-promoting cytokine, Interleukin-15 (IL-15), is often claimed to be a key pathogenic cytokine in alopecia areata (AA). Yet, rhIL-15 promotes human hair follicle (HF) growth ex vivo. We have asked whether the expression of IL-15 and its receptor (IL-15R) isoforms is altered in human AA and how IL-15 impacts on human HF immune privilege (HF-IP) in the presence/absence of interferon-γ (IFNγ), the well-documented key AA-pathogenic cytokine, as well as on hair regrowth after experimental AA induction in vivo. Quantitative immunohistomorphometry showed the number of perifollicular IL-15+ T cells in AA skin biopsies to be significantly increased compared to healthy control skin, while IL-15, IL-15Rα, and IL-15Rγ protein expression within the hair bulb were significantly down-regulated in AA HFs. In organ-cultured human scalp HFs, rhIL-15 significantly reduced hair bulb expression of MICA, the key “danger” signal in AA pathogenesis, and increased production of the HF-IP guardian, α-MSH. Crucially, ex vivo, rhIL-15 prevented IFNγ-induced HF-IP collapse, restored a collapsed HF-IP by IL-15Rα-dependent signaling (as documented by IL-15Rα−silencing), and protected AA-preventive immunoinhibitory iNKT10 cells from IFNγ-induced apoptosis. rhIL-15 even promoted hair regrowth after experimental AA induction in human scalp skin xenotransplants on SCID/beige mice in vivo. Our data introduce IL-15 as a novel, functionally important HF-IP guardian whose signaling is constitutively defective in scalp HFs of AA patients. Our data suggest that selective stimulation of intrafollicular IL-15Rα signaling could become a novel therapeutic approach in AA management, while blocking it pharmacologically may hinder both HF-IP restoration and hair re-growth and may thus make HFs more vulnerable to AA relapse.

Mechanical epilation exerts complex biological effects on human hair follicles and perifollicular skin: An exvivo study approach.

Electrical epilation of unwanted hair is a widely used hair removal method, but it is largely unknown how this affects the biology of human hair follicles (HF) and perifollicular skin. Here, we have begun to explore how mechanical epilation changes selected key biological read-out parameters exvivo within and around the pilosebaceous unit. Human full-thickness scalp skin samples were epilated exvivo using an electro-mechanical device, organ-cultured for up to 6 days in serum-free, supplemented medium, and assessed at different time points by quantitative (immuno-)histomorphometry for selected relevant read-out parameters in epilated and sham-epilated control samples. Epilation removed most of the hair shafts, often together with fragments of the outer and inner root sheath and hair matrix. This was associated with persistent focal thinning of the HF basal membrane, decreased melanin content of the residual HF epithelium, and increased HF keratinocyte apoptosis, including in the bulge, yet without affecting the number of cytokeratin 15+ HF epithelial stem cells. Sebocyte apoptosis in the peripheral zone was increased, albeit without visibly altering sebum production. Epilation transiently perturbed HF immune privilege, and increased the expression of ICAM-1 in the bulge and bulb mesenchyme, and the number of perifollicular MHC class II+ cells as well as mast cells around the distal epithelium and promoted mast cell degranulation around the suprabulbar and bulbar area. Moreover, compared to controls, several key players of neurogenic skin inflammation, itch, and/or thermosensation (TRPV1, TRPA1, NGF, and NKR1) were differentially expressed in post-epilation skin. These data generated in denervated, organ-cultured human scalp skin demonstrate that epilation-induced mechanical HF trauma elicits surprisingly complex biological responses. These may contribute to the delayed re-growth of thinner and lighter hair shafts post-epilation and temporary post-epilation discomfort. Our findings also provide pointers regarding the development of topically applicable agents that minimize undesirable sequelae of epilation.

The co-expression pattern of VEGFR-2 with indicators related to proliferation, apoptosis, and differentiation of anagen hair follicles.

An increasing number of studies show that vascular endothelial growth factor is an important regulator of hair growth, and involves in processes of hair follicle development by vascularization. Recently, VEGF receptor-2 (VEGFR-2) has been detected in epithelial cells of hair follicles, indicating that it may have a direct role in the biological activity of hair follicles. To explore how VEGFR-2 regulates hair follicle development, we investigated the co-expression pattern of VEGFR-2 with β-catenin, Bax, Bcl-2, involucrin, AE13 (hair cortex cytokeratin), keratin 16, keratin 14, and Laminin 5 by immunofluorescence double staining in anagen hair follicles of normal human scalp skin. The results of double staining immunofluorescence showed a strong overlapping and similar expression pattern for VEGFR-2 with β-catenin and Bcl-2, and revealing associated expression pattern with involucrin, AE13, keratin 14, keratin 16, and Laminin 5. These results elucidated that VEGFR-2 activation may participate in hair follicle differentiation, proliferation, and apoptosis in vivo.

Involvement of ILC1-like innate lymphocytes in human autoimmunity, lessons from alopecia areata.

Here, we have explored the involvement of innate lymphoid cells-type 1 (ILC1) in the pathogenesis of alopecia areata (AA), because we found them to be significantly increased around lesional and non-lesional HFs of AA patients. To further explore these unexpected findings, we first co-cultured autologous circulating ILC1-like cells (ILC1lc) with healthy, but stressed, organ-cultured human scalp hair follicles (HFs). ILClc induced all hallmarks of AA ex vivo: they significantly promoted premature, apoptosis-driven HF regression (catagen), HF cytotoxicity/dystrophy, and most important for AA pathogenesis, the collapse of the HFs physiological immune privilege. NKG2D-blocking or IFNγ-neutralizing antibodies antagonized this. In vivo, intradermal injection of autologous activated, NKG2D+/IFNγ-secreting ILC1lc into healthy human scalp skin xenotransplanted onto SCID/beige mice sufficed to rapidly induce characteristic AA lesions. This provides the first evidence that ILC1lc, which are positive for the ILC1 phenotype and negative for the classical NK markers, suffice to induce AA in previously healthy human HFs ex vivo and in vivo, and further questions the conventional wisdom that AA is always an autoantigen-dependent, CD8 +T cell-driven autoimmune disease.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Latanoprost-Loaded Nanotransfersomes Designed for Scalp Administration Enhance Keratinocytes Proliferation.

Latanoprost (LAT) has been shown to have a hypertrichotic effect, which makes it a promising candidate for alopecia treatments. For the first time, LAT has been encapsulated in nanotransfersomes in order to increase its efficacy. Ex vivo skin biodistribution was studied by confocal laser microscopy both in human scalp and pig skin. Results showed that nanotransfersomes increase the penetration of two different fluorochromes, with similar patterns in both species, compared with fluorochrome solutions containing no nanotransfersomes. Nanotransfersomes were stable under accelerated conditions (40 °C/75% RH) and long-term conditions (25 °C/60% RH) for up to 1 year, with no differences in vesicle size and polydispersity when LAT was loaded. Nanotransfersomes increased the LAT cell proliferation effect in HaCaT cell via MAPK signaling pathway. Collectively, our results demonstrate LAT-nanotransfersomes formulation could be a promising therapy for hair growth disorders.

014 Tyrosine Kinase 2 Inhibition Ameliorates the Phenotype of Lesional Alopecia Areata Scalp Skin Ex Vivo, and Reverses the Induction of Human Alopecia Areata in a Humanized Mouse Model

Alopecia areata (AA) is an immune-mediated hair follicle (HF) disorder induced by elevated IFNγ levels and a Tc1-driven inflammatory response. We have previously shown that treatment with IL-12 and IL-18 induces IFNγ-dependent, immune privilege collapse of the hair bulb, which can be inhibited by blocking IL-12 receptor signaling with the TYK2 inhibitor, BMS-986202 (BMS), in human microdissected HFs. Here, we further investigate the role of TYK2 and its inhibition in AA pathogenesis. We cultured human full thickness non-lesional and lesional scalp skin biopsies from acute or chronic AA patients ex vivo with BMS.

732 Treatment with cyclohexyl salicylate, an olfactory receptor 2A4/7 agonist, promotes human hair follicle growth and bulge stem cell progeny expansion

Topically applicable non-drugs that enhance the efficacy of available FDA-licensed drugs would be a welcome therapeutic addition in hair loss management. Since the olfactory receptor (OR) agonist, Sandalore, promotes human hair growth and reduces telogen effluvium, we have explored here whether other cosmetic OR ligands can unfold similar properties. We focused on OR2A4/7, since its stimulation promotes epidermal keratinocyte proliferation and differentiation. In situ hybridization showed that OR2A4/7 mRNA is widely expressed in the hair follicle (HF) epithelium, namely the outer root sheath (ORS) and hair matrix (HM). Yet, in freshly embedded human scalp skin, OR2A4/7 protein was mainly restricted to the infundibulum. However, OR2A4/7 protein became widely expressed in the ORS and HM during HF organ culture (OC), suggesting up-regulation of intrafollicular OR2A4/7 expression under tissue stress conditions. In scalp HF OC, the cosmetic OR2A4/7 agonist, cyclohexyl salicylate (CHS), delayed catagen development and tendentially increased HM proliferation. While CHS did not seem to impact on K15+ bulge stem cells, the % of CD34+ cells, i.e. their immediate progeny, was significantly increased in the suprabulbar ORS, as was the % of CD71+ transit amplifying cells (thought to derive from CD34+ cells) in the HM and suprabulbar ORS. Thus, stimulating OR2A4/7 via the non-drug CHS promotes hair growth and expands the progeny of K15+ stem cells, inviting the use of CHS as a novel cosmetic adjuvant treatment for hair loss disorders characterized by premature catagen development and a reduced capacity of K15+ stem cells to generate progeny, such as androgenetic alopecia.

342 Human skin and its appendages regeneration in the large full-thickness xenografting model

Most of the skin biology data obtained from a mouse model. However, studies of human skin have shown differences in the cell molecular signatures, the processes of physiological regeneration, wound healing, the development of diseases, etc. Human skin xenografting to immunodeficiency mice allows the most complex experiments to study normal and pathological processes, the search for methods of therapy for various diseases of human skin. The main limitation of this approach is the graft size. Most studies use partial-thickness xenografts without terminal hairs with excision of the dermal part along with adipose tissue while small punch biopsies or follicular units of human scalp skin are used to study the structure of hair follicles. The purpose of the study was to develop a xenografting model of a reasonably large full-thickness skin flap containing terminal hair follicles. The graft size was 15×5 mm. The skin was successfully engrafted, all skin structures including epidermis, dermis, blood vessels, hair follicles, sebaceous and sweat glands were preserved. The xenografting procedure is traumatic and promotes catagen induction. On day 32, we observed active proliferation of the graft tissue, a network of small vessels in the dermis and epidermal ridges at the junction of the graft with the recipient’s skin. On day 78, the xenograft was at the late stages of regeneration, there was a general decrease in proliferation with reconstruction of papillary and reticular dermis, and hair follicles morphology corresponded to the anagen stage. On day 110, visible growth of hair shafts above the skin surface was observed. Our data indicate that the homeostasis establishment in a 15×5 mm full-thickness skin xenograft with terminal hair follicles is longer than that in a split-thickness skin graft and takes more than 12 weeks. The described humanized skin model is suitable for studying the regeneration of skin and its appendages with surrounding tissues.

Resident human dermal γδT-cells operate as stress-sentinels: Lessons from the hair follicle

Murine γδT-cells have stress-surveillance functions and are implicated in autoimmunity. Yet, whether human γδT-cells are also stress sentinels and directly promote autoimmune responses in the skin is unknown. Using a novel (mini-)organ assay, we tested if human dermis resident γδT-cells can recognize stressed human scalp hair follicles (HFs) to promote an alopecia areata (AA)-like autoimmune response. Accordingly, we show that γδT-cells from healthy human scalp skin are activated (CD69+), up-regulate the expression of NKG2D and IFN-γ, and become cytotoxic when co-cultured with autologous stressed HFs ex vivo. These autologous γδT-cells induce HF immune privilege collapse, dystrophy, and premature catagen, i.e. three hallmarks of the human autoimmune HF disorder, AA. This is mediated by CXCL12, MICA, and in part by IFN-γ and CD1d. In conclusion, human dermal γδT-cells exert physiological stress-sentinel functions in human skin, where their excessive activity can promote autoimmunity towards stressed HFs that overexpress CD1d, CXCL12, and/or MICA.

LB709 Therapeutic effect of γδTregs cells in Alopecia Areata

Regulatory γδ T cells (γδTregs) may exert therapeutic effects under some experimental autoimmune disease (AID) conditions. This encouraged us to explore whether this this also applies to alopecia areata (AA), one of the most common human AIDs. Triple immunofluorescence (IF) staining was performed to determine the distribution of γδTregs in human skin presence of γδTregs on skin sections of AA patients. Autologous circulating human γδ Tregs were expanded and either co-cultured with organ-cultured, stressed human scalp hair follicles (HFs) ex vivo or injected into experimentally induced AA lesions on human scalp skin xenotransplants on SCID mice.

Prediction of the Risk of Alopecia Areata Progressing to Alopecia Totalis and Alopecia Universalis: Biomarker Development with Bioinformatics Analysis and Machine Learning

Background: Alopecia areata (AA) is an autoimmune disease typified by nonscarring hair loss with a variable clinical course. Although there is an increased understanding of AA pathogenesis and progress in its treatments, the outcome of AA patients remains unfavorable, especially when they are progressing to the subtypes of alopecia totalis (AT) or alopecia universalis (AU). Thus, identifying biomarkers that reflect the risk of AA progressing to AT or AU could lead to better interventions for AA patients. Methods: In this study, we conducted bioinformatics analyses to select key genes that correlated to AU or AT based on the whole-genome gene expression of 122 human scalp skin biopsy specimens obtained from NCBI-GEO GSE68801. Then, we built a biomarker using 8 different machine learning (ML) algorithms based on the key genes selected by bioinformatics analyses. Results: We identified 4 key genes that significantly increased (CD28) or decreased (HOXC13, KRTAP1-3, and GPRC5D) in AA tissues, especially in the subtypes of AT and AU. Besides, the predictive accuracy (area under the curve [AUC] value) of the prediction models for forecasting AA patients progressing to AT/AU models reached 90.7% (87.9%) by logistic regression, 93.8% (79.9%) by classification trees, 100.0% (76.3%) by random forest, 96.9% (76.3%) by support vector machine, 83.5% (79.9%) by K-nearest neighbors, 97.1% (87.3%) by XGBoost, and 93.3% (80.6%) by neural network algorithms for the training (internal validation) cohort. Besides, 2 molecule drugs, azacitidine and anisomycin, were identified by Cmap database. They might have the potential therapeutic effects on AA patients with high risk of progressing to AT/AU. Conclusions: In the present study, we conducted high accuracy models for predicting the risk of AA patients progressing to AT or AU, which may be important in facilitating personalized therapeutic strategies and clinical management for different AA patients.

Pro-inflammatory Vδ1+T-cells infiltrates are present in and around the hair bulbs of non-lesional and lesional alopecia areata hair follicles

BackgroundIt is widely accepted that NKG2D+cells are critically involved in alopecia areata (AA) pathogenesis. However, besides being expressed in CD8+T-cells and NK cells, NKG2D is also found in human γδT-cells. AA lesional hair follicles (HFs) overexpress NKG2D and γδTCR activating ligands, e.g. MICA and CD1d, and chemoattractants for γδT-cells, such as CXCL10. ObjectiveTo investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis. MethodsWe analyzed the number and activation status of γδT-cells in human healthy, lesional and non-lesional AA scalp biopsies by FACS and/or quantitative (immuno-)histomorphometry. ResultsIn healthy human scalp skin, the few skin-resident γδT-cells were found to be mostly Vδ1+, non-activated (CD69−NKG2Ddim) and positive for CXCL10, and CXCL12 receptors. These Vδ1+T-cells predominantly localized in/around the HF infundibulum. In striking contrast, the number of Vδ1+T-cells was significantly higher around and even inside the proximal (suprabulbar and bulbar) epithelium of lesional AA HFs. These cells also showed a pro-inflammatory phenotype, i.e. higher NKG2D, and IFN-γ and lower CD200R expression. Importantly, more pro-inflammatory Vδ1+T-cells were seen also around non-lesional AA HFs. Lesional AA HFs also showed significantly higher expression of CXCL12. ConclusionOur pilot study introduces skin-resident γδT-cells as a previously overlooked, but potentially important, mostly (auto-)antigen-independent, new innate immunity protagonist in AA pathobiology. The HF infiltration of these activated, IFN-γ-releasing cells already around non-lesional AA HFs suggest that Vδ1+T-cells are involved in the early stages of human AA pathobiology, and may thus deserve therapeutic targeting for optimal AA management.

Hormonal Effects on Hair Follicles.

The hair cycle and hair follicle structure are highly affected by various hormones. Androgens—such as testosterone (T); dihydrotestosterone (DHT); and their prohormones, dehydroepiandrosterone sulfate (DHEAS) and androstendione (A)—are the key factors in terminal hair growth. They act on sex-specific areas of the body, converting small, straight, fair vellus hairs into larger darker terminal hairs. They bind to intracellular androgen receptors in the dermal papilla cells of the hair follicle. The majority of hair follicles also require the intracellular enzyme 5-alpha reductase to convert testosterone into DHT. Apart from androgens, the role of other hormones is also currently being researched—e.g., estradiol can significantly alter the hair follicle growth and cycle by binding to estrogen receptors and influencing aromatase activity, which is responsible for converting androgen into estrogen (E2). Progesterone, at the level of the hair follicle, decreases the conversion of testosterone into DHT. The influence of prolactin (PRL) on hair growth has also been intensively investigated, and PRL and PRL receptors were detected in human scalp skin. Our review includes results from many analyses and provides a comprehensive up-to-date understanding of the subject of the effects of hormonal changes on the hair follicle.

Correction to: Enhanced Follicular Delivery of Finasteride to Human Scalp Skin Using Heat and Chemical Penetration Enhancers.

The article Enhanced Follicular Delivery of Finasteride to Human Scalp Skin Using Heat and Chemical Penetration Enhancers, written by Farah, Brown, and McAuley was originally published electronically on the publisher's internet portal on 31 May, 2020 without open access.

Enhanced Follicular Delivery of Finasteride to Human Scalp Skin Using Heat and Chemical Penetration Enhancers

PurposeThe aim of this work was to evaluate whether improved topical delivery of finasteride, focussed to the hair follicles of human scalp skin could be achieved with application of short durations of heat and use of specific chemical penetration enhancers.MethodsFranz cell experiments with human scalp skin were performed with a range of chemical penetration enhancers at 32°C and 45°C to simulate normal and heated conditions. Selected chemical penetration enhancers were taken forward for finite dose Franz cell studies which examined the effect of heat produced by a prototype external heating system that supplied either 20 or 30 min of additional heat over both a 24 h and a 1 h time period.ResultsShort durations of externally applied heat significantly increased finasteride penetration into human scalp skin after 24 h. Analysis of drug distribution in the skin after 1 h and 24 h indicated that both heat and chemical penetration enhancer selection influenced drug delivery to the hair follicles.ConclusionThe use of short durations of heat in combination with specific chemical penetration enhancers was able to increase the delivery of finasteride to human scalp skin and provide focussed drug delivery to the hair follicles.

Effect of Chrysanthemum zawadskii Extract on Dermal Papilla Cell Proliferation and Hair Growth.

BackgroundChrysanthemum zawadskii (CZ) belongs to the genus Chrysanthemum, also known as ‘Gu-Jeol-Cho’ in Korea. CZ has been used as herbal remedy to manage cough, hypertensive disorders, pharyngitis, bronchitis, gastroenteritis, pneumonia, bladder diseases and common cold. However, its effect on hair growth has not been documented.ObjectiveThe aim of present study was to elucidate the beneficial effects of CZ on hair growth.MethodsProliferation of follicular dermal papilla (DP) cells from human scalp skin was evaluated by MTT assay. The expression of various molecules in DP cells was checked by western blot assay. Effect of CZ extract on the hair growth was evaluated by hair organ culture and C57BL/6 mice model.ResultsCultivation of DP cells with CZ extract increased cellular proliferation, increased expression of phosphorylated protein kinase B (p-Akt), p-ERK, B-cell lymphoma 2, and decreased expression of Bax. Treatment of human hair follicles with CZ extract significantly enhanced hair growth. Additionally, CZ markedly shortened telogen period, increased anagen transformation and stimulated hair growth in the animal study.ConclusionThese results suggest that CZ extract has an effect of promoting hair growth and may therefore be a useful a therapeutic remedy for preventing hair loss.

An osteopontin-derived peptide inhibits human hair growth at least in part by decreasing fibroblast growth factor-7 production in outer root sheath keratinocytes.

Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. Our hypothesis was tested exvivo and invivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L-1 to 3 μmol L-1 ) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth exvivo and invivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic? The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add? We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) exvivo and invivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both exvivo and invivo. What is the translational message? High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth.

Effects of the selective TrkA agonist gambogic amide on pigmentation and growth of human hair follicles in vitro.

The human hair follicle is a neuroendocrine mini-organ that can be used to study aging processes in vitro. Neurotrophins maintain homeostasis in hair biology via the Trk-family of receptors. TrkA, the high affinity receptor for nerve growth factor (NGF), is expressed in hair follicle melanocytes and keratinocytes, where it regulates proliferation, differentiation and apoptosis and may thereby play a role in hair pigmentation and growth. We investigated TrkA expression during the human hair cycle and the effects of a selective high affinity TrkA agonist, Gambogic Amide, on hair pigmentation and hair growth in human hair follicles in vitro. In human scalp skin, TrkA expression was strongest in proliferating melanocytes re-establishing the pigmentary unit in the hair bulb during the early hair growth phase, anagen. During high anagen and in the de-composing pigmentary-unit of the regression phase, catagen, bulb-melanocytes lost TrkA expression and only undifferentiated outer root sheath melanocytes maintained it. In cultured human anagen hair follicles, Gambogic Amide was able to prevent gradual pigment loss, while it stimulated hair shaft elongation. This was achieved by increased melanocyte activation, migration and dendricity, highlighted by distinct c-KIT-expression in melanocyte sub-populations. Our results suggest that Gambogic Amide can maintain hair follicle pigmentation by acting on undifferentiated melanocytes residing in the outer root sheath and making them migrate to establish the pigmentary-unit. This suggests that the selective TrkA agonist Gambogic Amide acts as an anti-hair greying and hair growth promoting molecule in vitro.