Topical tacrolimus suppresses the expression of vascular endothelial growth factor and insulin‐like growth factor‐1 in late anagen

Tacrolimus has shown promising results in the treatment of various dermatological diseases, including hair loss. The direct effect of tacrolimus on hair follicles and its underlying mechanisms have rarely been investigated. In this study, we investigated the effects of topical tacrolimus on anagen in the hair cycle and on the expression of vascular endothelial growth factor (VEGF) and insulin‐like growth factor‐1 (IGF‐1) mRNAs in mouse skin. Topical tacrolimus 0.1% ointment was applied to one side of the skin of depilated C57BL/6 mice. Skin samples from both sides were taken during the study. Vegf and Igf‐1 mRNA were determined by quantitative RT‐PCR. No obvious difference in skin colour, hair cycling or histological features was found between the treated and untreated skin, but the levels of Vegf mRNA and Igf‐1 mRNA were markedly decreased in the treated skin in late anagen, compared with those in untreated skin.     

Evidence that the bulge region is a site of relative immune privilege in human hair follicles

Background Recent gene profiling data suggest that, besides the anagen hair bulb, the epithelial stem cell region in the outer root sheath of hair follicles (HFs), termed the bulge, may also represent an area of relative immune privilege (IP). Objectives To investigate whether the human HF bulge is a site of relative IP within anagen VI HFs. Methods Anagen VI HFs from normal human scalp skin were analysed using immunohistological staining techniques, quantitative histomorphometry and statistical analysis. For functional evidence we performed full‐thickness human scalp skin organ cultures to investigate whether interferon (IFN)‐γ, a key inducer of IP collapse in hair bulbs, has a similar effect on the putative bulge IP. Results Major histocompatibility complex (MHC) class Ia, β₂‐microglobulin and MHC class II immunoreactivity are downregulated in the human bulge. The immunosuppressants α‐melanocyte stimulating hormone, transforming growth factor‐β2, macrophage migration inhibitory factor and indoleamine‐2,3‐dioxygenase (IDO) are upregulated in the CD200+, stem cell‐rich bulge region. These CD200+ cells also co‐express HLA‐E. Furthermore, IFN‐γ induces significant ectopic MHC class Ia expression in bulge cells of organ‐cultured human scalp skin. Conclusions These data suggest that the bulge of human anagen HFs represents a hitherto unrecognized site of relative IP in human skin. Simultaneously, we present the first evidence of IDO and HLA‐E protein expression in normal human HFs. Bulge IP presumably protects the HF epithelial stem cell reservoir from autoaggressive immune attack whereas a loss of bulge IP may play a central role in the pathogenesis of cicatricial alopecias.     

Injury modifies the fate of hair follicle dermal stem cell progeny in a hair cycle‐dependent manner

The dermal papilla (DP) is one of two principal mesenchymal compartments of the hair follicle (HF). We previously reported that a population of HF dermal stem cells (hfDSCs) function to regenerate the dermal sheath (DS), but intriguingly also contribute new cells to the adult DP at the onset of anagen hair growth to maintain normal cycling of HFs and support the production of large hair fibres. Here, we asked whether injury altered the behaviour of hfDSCs and their progeny in order to support wound‐induced hair growth (WIHG) and if the response was modulated by hair cycle stage. αSMACreER^T2:ROSA^YFP mice received tamoxifen to label the DS, including hfDSCs. Full‐thickness excisions were made on the dorsal skin during various stages of the hair cycle. The skin was harvested at the subsequent anagen. Interestingly, there was an increase in the magnitude of recruitment of hfDSC progeny into the DP after injury compared to follicles entering natural second anagen. This bias towards a DP fate only occurred when a wound was induced during certain stages of the HC. In summary, injury modifies the fate of hfDSCs progeny, biasing them towards recruitment into the DP, with the hair cycle stage also influencing this response.     

Intact hair follicle innervation is not essential for anagen induction and development

Abstract Neuropeptides produced, stored and secreted by the unusually dense sensory and autonomic innervation of hair follicles (HFs) can induce hair growth (anagen) and may be involved in hair growth control. To test the role of follicle innervation of HF cycling in vivo, we generated innervation-deficient HFs by unilateral surgical denervation of a defined region of back skin in C57BL/6 mice and assessed its effect on spontaneous and induced anagen development. Successful denervation was demonstrated by the absence of PGP 9.5+ or tyrosine hydroxylase+ nerves and nerve-associated neuropeptides (substance P, CGRP). By quantitative histomorphometry, no significant difference in spontaneous or cyclosporin A-induced anagen development could be detected between sham-operated control skin and denervated skin. Only after hair growth induction by depilation, a discrete, marginally significant retardation of anagen development was apparent in denervated HFs. Thus, even though cutaneous nerves may exert a minor modulatory role in depilation-induced hair growth, they are not essential for normal murine anagen development. Received: 4 March 1998     

Comparative regenerative biology of spiny (Acomys cahirinus) and laboratory (Mus musculus) mouse skin

Wound‐induced hair follicle neogenesis (WIHN) has been demonstrated in laboratory mice (Mus musculus) after large (>1.5 × 1.5 cm²) full‐thickness wounds. WIHN occurs more robustly in African spiny mice (Acomys cahirinus), which undergo autotomy to escape predation. Yet, the non‐WIHN regenerative ability of the spiny mouse skin has not been explored. To understand the regenerative ability of the spiny mouse, we characterized skin features such as hair types, hair cycling, and the response to small and large wounds. We found that spiny mouse skin contains a large portion of adipose tissue. The spiny mouse hair bulge is larger and shows high expression of stem cell markers, K15 and CD34. All hair types cycle synchronously. To our surprise, the hair cycle is longer and less frequent than in laboratory mice. Newborn hair follicles in anagen are more mature than C57Bl/6 and demonstrate molecular features similar to C57Bl/6 adult hairs. The second hair cycling wave begins at week 4 and lasts for 5 weeks, then telogen lasts for 30 weeks. The third wave has a 6‐week anagen, and even longer telogen. After plucking, spiny mouse hairs regenerate in about 5 days, similar to that of C57Bl/6. After large full‐thickness excisional wounding, there is more de novo hair formation than C57Bl/6. Also, all hair types are present and pigmented, in contrast to the unpigmented zigzag hairs in C57Bl/6 WIHN. These findings shed new light on the regenerative biology of WIHN and may help us understand the control of skin repair vs regeneration.     

Modulating hair follicle size with Wnt10b/DKK1 during hair regeneration

Hair follicles have characteristic sizes corresponding to their cycle‐specific stage. However, how the anagen hair follicle specifies its size remains elusive. Here, we showed that in response to prolonged ectopic Wnt10b‐mediated β‐catenin activation, regenerating anagen hair follicles grew larger in size. In particular, the hair bulb, dermal papilla and hair shaft became enlarged, while the formation of different hair types (Guard, Awl, Auchene and Zigzag) was unaffected. Interestingly, we found that the effect of exogenous WNT10b was mainly on Zigzag and less on the other kinds of hairs. We observed dramatically enhanced proliferation within the matrix, DP and hair shaft of the enlarged AdWnt10b‐treated hair follicles compared with those of normal hair follicles at P98. Furthermore, expression of CD34, a specific hair stem cell marker, was increased in its number to the bulge region after AdWnt10b treatment. Ectopic expression of CD34 throughout the ORS region was also observed. Many CD34‐positive hair stem cells were actively proliferating in AdWnt10b‐induced hair follicles. Importantly, subsequent co‐treatment with the Wnt inhibitor, DKK1, reduced hair follicle enlargement and decreased proliferation and ectopic localization of hair stem cells. Moreover, injection of DKK1 during early anagen significantly reduced the width of prospective hairs. Together, these findings strongly suggest that Wnt10b/DKK1 can modulate hair follicle size during hair regeneration.     

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15‐positive hair bulge epithelium of both anagen‐ and telogen‐phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.     

CRH peptides modulate human hair follicle growth in vitro– a preliminary study

Mammalian skin may contain an equivalent of the hypothalamic‐pituitary‐adrenal axis (HPA), composed of locally produced corticotropin‐releasing hormone (CRH) that, together with signalling via CRH receptor 1 (CRH‐R1) and CRH‐R2, may regulate local homeostasis. Studies in murine skin have demonstrated significant hair cycle‐dependent fluctuations in the expression of CRH and urocortin peptides and CRH‐Rs genes, suggesting a modulatory role for this signalling system in hair growth/cycling. This study was designed to investigate the effects of ligands showing increased selectivity for CRH‐R1 [(D‐Glu²⁰)‐CRH (10^?7 and 10^?8 m )] and CRH‐R2 [(D‐Pro⁵)‐CRH (10^?8 and 10^?9 m )] and (D‐Pro⁴)‐urocortin (10^?7 and 10^?8 m ) on human hair growth in ex vivo culture. (D‐Pro⁵)‐CRH can also activate CRH‐R1, while (D‐Pro⁴)‐urocortin is highly selective for CRH‐R2. Anagen hair follicles (HFs) were isolated from human scalp and stimulated for 9 days, with 10 HFs tested per CRH peptide concentration. Preliminary findings indicate that (D‐Pro⁴)‐urocortin (10^?8 m ) stimulated a 79% mean hair fibre elongation compared to the initial HF length over the 9‐day study period. (D‐Glu²⁰)‐CRH (10^?8 m ) also stimulated hair fibre elongation of 63% of initial length, while (D‐Pro⁵)‐CRH (10^?9 m ), which inhibited hair fibre elongation, compared to unstimulated controls. In agreement with our previous detection of CRH‐R1 and CRH‐R2 in human HFs, the above data suggest the existence of a functionally active CRH peptide/receptor system in cultured human HFs and suggest that signalling via these receptors may participate in the regulation of human hair growth/cycling in vivo.     

Mammalian target of rapamycin complex 1 (mTORC1) may modulate the timing of anagen entry in mouse hair follicles

Mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and survival. There is limited evidence that mTOR influences hair follicles (HFs), which undergo cycles of quiescence (telogen), growth (anagen) and regression (catagen). We sought to investigate whether mTOR, in particular mTOR complex 1 (mTORC1), regulates the hair growth cycle by employing biochemical, immunohistochemical and functional approaches in vivo. Here, we demonstrate that quantitative analysis of mTORC1 kinase activity shows phase‐dependent changes, and phosphorylated mTOR at S2448 (p‐mTOR) was localized in certain sites of HFs in a phase‐dependent manner. These results were indicative of mTOR's role in hair growth initiation. Finally, in a pharmacological challenge in vivo using the specific mTORC1 inhibitor, rapamycin, hair cycle initiation was delayed, suggesting a functional relevance of mTORC1 in anagen entry. Based on our findings, we propose that mTORC1 may participate in hair cycle regulation, namely the timing of anagen initiation.     

Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture

Please cite this paper as: Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Experimental Dermatology 2010; 19: 305–312. Abstract: The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth‐modulatory effects of agents in this assay, one critical read‐out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ‐cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.     

Hair type‐specific function of canonical Wnt activity in adult mouse skin

Wnt/β‐catenin signalling is a key regulator of hair follicle (HF) morphogenesis and life‐long HF regeneration. In a recently published issue of Experimental Dermatology, Lei et al. report that sustained WNT10B supply and pathway activation in regenerating mouse HF increased the width of hair bulbs, hair shafts and the dermal papilla (DP), and enlarged the CD34⁺ HF bulge cell compartment. Notably, WNT10B affected primarily zigzag HFs, while size and morphology of other HF types remained largely unaffected. Thus, these findings raise a number of questions regarding a HF type‐specific function of Wnt/β‐catenin and on the role of the WNT‐stimulated DP in this process.     

Role of bulge cells in wound healing: possible implications for hidradenitis suppurativa

The hair follicle bulge harbors a cluster of quiescent epithelial stem cells, which generate the new lower hair follicle during follicle cycling. The role of bulge cells for maintenance of the hair follicle and epidermis during normal homeostasis and after wounding remains controversial. To address these questions, we targeted the suicide gene, thymidine kinase (TK), or CrePR recombinase to hair follicle bulge cells using a K15 promoter. Administration of ganciclovir to K15‐TK mice caused their death due to gastrointestinal injury. We then treated immunodeficient mice carrying K15‐TK skin grafts with ganciclovir. This resulted in injury of bulge cells within several days. Loss of hair and permanent injury to hair follicles ensued. Dermal scarring was also present. The epidermis without follicles survived for several months. To further assess the contribution of bulge cells to epidermis, we used double transgenic K15‐crePR1, R26R mice. After 5 days of treatment with RU‐486 (time 0), bulge cells and a small fraction of epidermal cells expressed LacZ under control of the ROSA promoter. To determine whether bulge cells moved into the epidermis under normal homeostatic conditions, we counted labeled epidermal basal cells at times 0, 30 days, and 180 days. We found that the percentage of labeled epidermal basal cells (approximately 1%) remained constant, indicating an absence of cell movement from the bulge to the epidermis. After punch wounding, bulge progeny were detected in a radial pattern in the re‐epithelialized area. Radial streaks of bulge cell progeny emanated from the hair follicles at the wound edge. Bulge cell progeny proliferated and expressed normal epidermal differentiation markers such as keratin 10 and loricrin. Overall, these results indicate that follicular stem cells in the bulge are necessary for hair follicle survival and that these cells functionally contribute to epidermal regeneration in response to wounding; however, the epidermis self‐renews autonomously of the bulge under normal conditions. The role of bulge cells in hidradenitis suppurativa (HS) is not known; however, pathological studies suggest aberrant proliferation of the hair follicle may accompany HS and inappropriate stem cell activation and differentiation could be a component of this disorder. The K15‐crePR1 transgenic mouse will serve as a powerful tool for evaluating the role of hair follicle stem cells in mouse models of HS. Studying hair follicle stem cells with recently defined markers for these cells will be useful for evaluating stem cell behavior in HS lesions.     

珊瑚姜提取物对C3H/He小鼠毛发生长周期及其对小鼠触须毛囊体外培养的影响

目的：探讨中药珊瑚姜对C3H／He小鼠毛发生长周期及其对小鼠触须毛囊体外培养的作用。方法：采用体外局部直接给药的方式，观察珊瑚姜提取物对C3H／He小鼠毛发生长的影响。结果：肉眼观察19d以后，2．0％珊瑚姜用药组小鼠毛已全部长满，0．5％珊瑚姜用药组小鼠也有长毛，较空白组多而长，毛色较空白对照组深，生长速度明显加快，提前进入生长期（P＜0．05）。病理切片显示用药15d后，用药组动物皮肤真皮层黑素分布较多，毛发处于生长期，而空白对照组黑素分布较少，毛发大多处于休止期。结论：珊瑚姜提取物具有明显的促毛发生长作用。     

The immune system of mouse vibrissae follicles: cellular composition and indications of immune privilege

Although vibrissae hair follicles (VHFs) have long been a key research model in the life sciences, their immune system (IS) is essentially unknown. Therefore, we have characterized basic parameters of the VHF‐IS of C57BL/6J mice by quantitative (immuno‐)histomorphometry. Murine anagen VHF harbour few CD4+ and CD8+ T cells in the distal mesenchyme and sinuses but hardly any gamma‐delta T cells in their distal epithelium. MHC class II+ Langerhans cells are seeded in the VHF infundibulum, which is also surrounded by MHC class II+ and CD11b+ cells (macrophages). The number of Langerhans cells then declines sharply in the VHF bulge, and the VHF bulb lacks MHC class II+ cells. Mast cells densely populate the VHF connective tissue sheath, where they strikingly cluster around the bulge. Both the bulge and the bulb of VHF display signs of immune privilege, that is, low MHC class I and MHC class II expression and local immunoinhibitor expression (CD200, TGFβ1). This immunophenotyping study fills an important gap in the immunobiology of murine skin and identifies differences between the IS of VHF, mouse pelage and human terminal HFs. This facilitates utilizing murine VHF as a versatile organ culture model for general immunology and immune privilege research in situ.     

Bleomycin‐induced fibrosis in MC1 signalling‐deficient C57BL/6J‐Mc1re/e mice further supports a modulating role for melanocortins in collagen synthesis of the skin

The melanocortin‐1 receptor (MC₁) binds α‐melanocyte‐stimulating hormone (α‐MSH) with high affinity and has a physiological role in cutaneous melanin pigmentation. Previously, we reported that human dermal fibroblasts also express functional MC₁. α‐MSH suppressed transforming growth factor‐β₁‐ and bleomycin (BLM)‐induced collagen synthesis in vitro and in vivo. Using MC₁ signalling‐deficient C57BL/6J‐Mc1r^e/e mice, we tested as to whether MC₁ has a regulatory role on dermal collagen synthesis in the BLM model of scleroderma. Notably, mice with a C57BL/6J genetic background were previously shown to be BLM‐non‐susceptible. Interestingly, treatment of C57BL/6J‐Mc1r^e/e but not of C57BL/6J‐wild‐type mice with BLM increased cutaneous collagen type I content at RNA and protein level along with development of skin fibrosis. Cutaneous levels of connective tissue growth factor and monocyte chemotactic protein‐1 were also increased in BLM‐treated C57BL/6J‐Mc1r^e/e mice. Primary dermal fibroblasts from C57BL/6J‐wt mice further expressed MC₁, suggesting that these cells are directly targeted by melanocortins to affect collagen production of the skin. Our findings support the concept that MC₁ has an endogenous regulatory function in collagen synthesis and controls the extent of fibrotic stress responses of the skin.     

Do human dermal adipocytes switch from lipogenesis in anagen to lipophagy and lipolysis during catagen in the human hair cycle?

In murine skin, dermal white adipose tissue (DWAT) undergoes fluctuations in size across the hair cycle, whereas changes in size, function and metabolism of dermal adipocytes (DAs) during the human scalp hair cycle remain unexplored. Transmission electron microscopy results suggest that during anagen‐catagen transition, human DAs co‐opt the autophagy machinery to undergo lipophagy within their lipid droplets. Whole‐mount staining of hair follicles (HFs) and surrounding DWAT for the autophagy marker LC3B confirms the increased presence of LC3B+ lipid droplets adjacent to catagen HFs; moreover, DWAT around catagen HFs engages in greater glycerol release compared to DWAT surrounding anagen HFs. Thus, we hypothesize that human DAs switch from lipogenesis during anagen to lipophagy together with lipolysis during catagen. We propose various experiments to further prove this hypothesis, whose systematic exploration should help to better characterize the functions of human DWAT and its communication with the HF.     

Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model

No model is available for examining whether in vivo‐damaged human hair follicles (hu‐HFs) are rescued by transplanting cultured hu‐HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo‐damaged hu‐HFs such as miniaturized hu‐HFs in androgenic alopecia are improvable by auto‐transplanting hu‐HF dermal cells. In this study, we first developed mice with humanized skin composed of hu‐keratinocytes and hu‐dermal fibroblasts. Then, a ‘humanized scalp model mouse’ was generated by transplanting hu‐scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu‐HFs in the model were amputated in situ, and cultured hu‐HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation.