Identifying novel strategies for treating human hair loss disorders: Cyclosporine A suppresses the Wnt inhibitor, SFRP1, in the dermal papilla of human scalp hair follicles.

Hair growth disorders often carry a major psychological burden. Therefore, more effective human hair growth-modulatory agents urgently need to be developed. Here, we used the hypertrichosis-inducing immunosuppressant, Cyclosporine A (CsA), as a lead compound to identify new hair growth-promoting molecular targets. Through microarray analysis we identified the Wnt inhibitor, secreted frizzled related protein 1 (SFRP1), as being down-regulated in the dermal papilla (DP) of CsA-treated human scalp hair follicles (HFs) ex vivo. Therefore, we further investigated the function of SFRP1 using a pharmacological approach and found that SFRP1 regulates intrafollicular canonical Wnt/ß-catenin activity through inhibition of Wnt ligands in the human hair bulb. Conversely, inhibiting SFRP1 activity through the SFRP1 antagonist, WAY-316606, enhanced hair shaft production, hair shaft keratin expression, and inhibited spontaneous HF regression (catagen) ex vivo. Collectively, these data (a) identify Wnt signalling as a novel, non-immune-inhibitory CsA target; (b) introduce SFRP1 as a physiologically important regulator of canonical ß-catenin activity in a human (mini-)organ; and (c) demonstrate WAY-316606 to be a promising new promoter of human hair growth. Since inhibiting SFRP1 only facilitates Wnt signalling through ligands that are already present, this 'ligand-limited' therapeutic strategy for promoting human hair growth may circumvent potential oncological risks associated with chronic Wnt over-activation.

Autophagy is essential for maintaining the growth of a human (mini-)organ: Evidence from scalp hair follicle organ culture.

Autophagy plays a crucial role in health and disease, regulating central cellular processes such as adaptive stress responses, differentiation, tissue development, and homeostasis. However, the role of autophagy in human physiology is poorly understood, highlighting a need for a model human organ system to assess the efficacy and safety of strategies to therapeutically modulate autophagy. As a complete, cyclically remodelled (mini-)organ, the organ culture of human scalp hair follicles (HFs), which, after massive growth (anagen), spontaneously enter into an apoptosis-driven organ involution (catagen) process, may provide such a model. Here, we reveal that in anagen, hair matrix keratinocytes (MKs) of organ-cultured HFs exhibit an active autophagic flux, as documented by evaluation of endogenous lipidated Light Chain 3B (LC3B) and sequestosome 1 (SQSTM1/p62) proteins and the ultrastructural visualization of autophagosomes at all stages of the autophagy process. This autophagic flux is altered during catagen, and genetic inhibition of autophagy promotes catagen development. Conversely, an anti-hair loss product markedly enhances intrafollicular autophagy, leading to anagen prolongation. Collectively, our data reveal a novel role of autophagy in human hair growth. Moreover, we show that organ-cultured scalp HFs are an excellent preclinical research model for exploring the role of autophagy in human tissue physiology and for evaluating the efficacy and tissue toxicity of candidate autophagy-modulatory agents in a living human (mini-)organ.

Hair growth control by innate immunocytes: Perifollicular macrophages revisited.

The role of innate immunocytes such as mast cells, γδ T cells, NK cells and macrophages (MACs) in hair growth control under physiological and pathological conditions has recently begun to be re-explored. Here, we revisit the role of resident perifollicular macrophages (pfMACs) located in the hair follicle (HF) mesenchyme (CTS). Substantial, stringently timed fluctuations in the number and localization of pfMACs were first observed long ago during murine HF morphogenesis and cycling. This already suggested some involvement of these innate immunocytes, with a recognized role in tissue remodelling and in hair growth control. The relatively recent demonstration of a Wnt signalling-driven crosstalk between these immunocytes and HF epithelial stem cells in telogen HFs, which promotes anagen induction, has reinvigorated interest in the role that pfMAC plays in hair biology. Besides the apoptosis-associated secretion of stem cell-activating Wnts and the differential secretion of HF-targeting growth factors such as FGF-5 and FGF5s from pfMACs, we also explore how MAC polarization, and thus function, may be influenced by the local metabolic and immune environment. Moreover, we examine how pfMACs may contribute to hair cycle-associated angiogenesis, vascular remodelling, HF immune privilege and immunopathology. On this basis, we discuss why targeting pfMACs may be relevant in the management of hair growth disorders. Finally, we argue that studying pfMACs offers an excellent, clinically relevant model system for characterizing and experimentally manipulating MAC interactions with an easily accessible mammalian, continuously remodelled (mini-)organ under both physiological and pathological conditions.

An efficient method for eccrine gland isolation from human scalp.

We describe a simple and efficient method to isolate eccrine sweat glands from the human scalp. This method is inspired by the hair graft harvesting method used in hair transplantation. Based on the recently described anatomical relationship between the scalp hair follicle and the eccrine gland, we have found that scalp follicular unit grafts are an excellent eccrine gland isolation source, especially for the coiled component. In order to make the gland visible for stereoscopic microdissection, the follicular units need to be previously stained with a vital dye like methylene blue or neutral red. The simplicity and efficiency of this isolation method should encourage further research into human eccrine sweat gland function which has always been hindered by the difficulty of gland isolation.

Is the eccrine gland an integral, functionally important component of the human scalp pilosebaceous unit?

The pilosebaceous unit (PSU) and the eccrine sweat gland (ESG) are classically described as completely independent skin appendages. However, careful inspection of scalp follicular units reveals that the secretory segment of the ESG spatially approximates the hair follicle in a position below the sebaceous gland and the insertion of the arrector pili muscle. Therefore, we propose here that, contrary to conventional wisdom, the PSU and the ESG should not be viewed in isolation, and may form instead, along with the arrector pili muscle and the apocrine gland (where present),one functional unit. For this, we suggest the more inclusive term of 'Hair Cluster' (HC). If confirmed, e.g. by 3D imaging techniques, the novel concept of a functional HC, whose individual components may communicate via secreted molecules and may share selected progenitor cell populations for HC repair/regeneration, has major physiological and pathological implications, which are briefly discussed.

Dermal Adipose Tissue Secretes HGF to Promote Human Hair Growth and Pigmentation.

Hair follicles (HFs) are immersed within dermal white adipose tissue (dWAT), yet human adipocyte‒HF communication remains unexplored. Therefore, we investigated how perifollicular adipocytes affect the physiology of human anagen scalp HFs. Quantitative immunohistomorphometry, X-ray microcomputed tomography, and transmission electron microscopy showed that the number and size of perifollicular adipocytes declined during anagen‒catagen transition, whereas fluorescence-lifetime imaging revealed increased lipid oxidation in adipocytes surrounding the bulge and/or sub-bulge region. Ex vivo, dWAT tendentially promoted hair shaft production, and significantly stimulated hair matrix keratinocyte proliferation and HF pigmentation. Both dWAT pericytes and PREF1/DLK1+ adipocyte progenitors secreted HGF during human HF‒dWAT co-culture, for which the c-Met receptor was expressed in the hair matrix and dermal papilla. These effects were reproduced using recombinant HGF and abrogated by an HGF-neutralizing antibody. Laser-capture microdissection‒based microarray analysis of the hair matrix showed that dWAT-derived HGF upregulated keratin (K) genes (K27, K73, K75, K84, K86) and TCHH. Mechanistically, HGF stimulated Wnt/ß-catenin activity in the human hair matrix (increased AXIN2, LEF1) by upregulating WNT6 and WNT10B, and inhibiting SFRP1 in the dermal papilla. Our study demonstrates that dWAT regulates human hair growth and pigmentation through HGF secretion, and thus identifies dWAT and HGF as important novel molecular and cellular targets for therapeutic intervention in human hair growth and pigmentation disorders.

Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo.

Besides monocyte (MO)-derived macrophages (MACs), self-renewing tissue-resident macrophages (trMACs) maintain the intracutaneous MAC pool in murine skin. Here, we have asked whether the same phenomenon occurs in human skin using organ-cultured, full-thickness skin detached from blood circulation and bone marrow. Skin stimulation ex vivo with the neuropeptide substance P (SP), mimicking neurogenic skin inflammation, significantly increased the number of CD68+MACs in the papillary dermis without altering intracutaneous MAC proliferation or apoptosis. Since intraluminal CD14+MOs were undetectable in the non-perfused dermal vasculature, new MACs must have differentiated from resident intracutaneous progenitor cells in human skin. Interestingly, CD68+MACs were often seen in direct cell-cell-contact with cells expressing both, the hematopoietic stem cell marker CD34 and SP receptor (neurokinin-1 receptor [NK1R]). These cell-cell contacts and CD34+cell proliferation were up-regulated in SP-treated skin samples. Collectively, our study provides the first evidence that resident MAC progenitors, from which mature MACs can rapidly differentiate within the tissue, do exist in normal adult human skin. That these NK1R+trMAC-progenitor cells quickly respond to a key stress-associated neuroinflammatory stimulus suggests that this may satisfy increased local MAC demand under conditions of wounding/stress.

Delayed Hair Follicle Morphogenesis and Hair Follicle Dystrophy in a Lipoatrophy Mouse Model of Pparg Total Deletion.

PPARγ regulates multiple aspects of skin physiology, including sebocyte differentiation, keratinocyte proliferation, epithelial stem cell survival, adipocyte biology, and inflammatory skin responses. However, the effects of its global deletion, namely of nonredundant key functions of PPARγ signaling in mammalian skin, are yet unknown because of embryonic lethality. Here, we describe the skin and hair phenotype of a whole-body PPARγ-null mouse (PpargΔ/Δ), obtained by preserving PPARγ expression in the placenta. PpargΔ/Δ mice exhibited total lipoatrophy and complete absence of sebaceous glands. Right after birth, hair follicle (HF) morphogenesis was transiently delayed, along with reduced expression of HF differentiation markers and of transcriptional regulators necessary for HF development. Later, adult PpargΔ/Δ mice developed scarring alopecia and severe perifollicular inflammation. Skin analyses in other models of lipodystrophy, AZIPtg/+ and Adipoq-Cretg/+Ppargfl/fl mice, coupled with skin graft experiments, showed that the early defects observed in hair morphogenesis were caused by the absence of adipose tissue. In contrast, the late alteration of HF cycle and appearance of inflammation were observed only in PpargΔ/Δ mice and likely were due to the lack sebaceous glands. Our findings underscore the increasing appreciation for the importance of adipose tissue-mediated signals in HF development and function.

Thyroxine differentially modulates the peripheral clock: lessons from the human hair follicle.

The human hair follicle (HF) exhibits peripheral clock activity, with knock-down of clock genes (BMAL1 and PER1) prolonging active hair growth (anagen) and increasing pigmentation. Similarly, thyroid hormones prolong anagen and stimulate pigmentation in cultured human HFs. In addition they are recognized as key regulators of the central clock that controls circadian rhythmicity. Therefore, we asked whether thyroxine (T4) also influences peripheral clock activity in the human HF. Over 24 hours we found a significant reduction in protein levels of BMAL1 and PER1, with their transcript levels also decreasing significantly. Furthermore, while all clock genes maintained their rhythmicity in both the control and T4 treated HFs, there was a significant reduction in the amplitude of BMAL1 and PER1 in T4 (100 nM) treated HFs. Accompanying this, cell-cycle progression marker Cyclin D1 was also assessed appearing to show an induced circadian rhythmicity by T4 however, this was not significant. Contrary to short term cultures, after 6 days, transcript and/or protein levels of all core clock genes (BMAL1, PER1, clock, CRY1, CRY2) were up-regulated in T4 treated HFs. BMAL1 and PER1 mRNA was also up-regulated in the HF bulge, the location of HF epithelial stem cells. Together this provides the first direct evidence that T4 modulates the expression of the peripheral molecular clock. Thus, patients with thyroid dysfunction may also show a disordered peripheral clock, which raises the possibility that short term, pulsatile treatment with T4 might permit one to modulate circadian activity in peripheral tissues as a target to treat clock-related disease.

The peripheral clock regulates human pigmentation.

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

A meeting of two chronobiological systems: circadian proteins Period1 and BMAL1 modulate the human hair cycle clock.

The hair follicle (HF) is a continuously remodeled mini organ that cycles between growth (anagen), regression (catagen), and relative quiescence (telogen). As the anagen-to-catagen transformation of microdissected human scalp HFs can be observed in organ culture, it permits the study of the unknown controls of autonomous, rhythmic tissue remodeling of the HF, which intersects developmental, chronobiological, and growth-regulatory mechanisms. The hypothesis that the peripheral clock system is involved in hair cycle control, i.e., the anagen-to-catagen transformation, was tested. Here we show that in the absence of central clock influences, isolated, organ-cultured human HFs show circadian changes in the gene and protein expression of core clock genes (CLOCK, BMAL1, and Period1) and clock-controlled genes (c-Myc, NR1D1, and CDKN1A), with Period1 expression being hair cycle dependent. Knockdown of either BMAL1 or Period1 in human anagen HFs significantly prolonged anagen. This provides evidence that peripheral core clock genes modulate human HF cycling and are an integral component of the human hair cycle clock. Specifically, our study identifies BMAL1 and Period1 as potential therapeutic targets for modulating human hair growth.