Activation of mitochondrial aldehyde dehydrogenase 2 promotes hair growth in human hair follicles.

INTRODUCTION: Hair loss is a common phenomenon associated with various environmental and genetic factors. Mitochondrial dysfunction-induced oxidative stress has been recognized as a crucial determinant of hair follicle (HF) biology. Aldehyde dehydrogenase 2 (ALDH2) mitigates oxidative stress by detoxifying acetaldehyde. This study investigated the potential role of ALDH2 modulation in HF function and hair growth promotion. OBJECTIVES: To evaluate the effects of ALDH2 activation on oxidative stress in HFs and hair growth promotion. METHODS: The modulatory role of ALDH2 on HFs was investigated using an ALDH2 activator. ALDH2 expression in human HFs was evaluated through in vitro immunofluorescence staining. Ex vivo HF organ culture was employed to assess hair shaft elongation, while the fluorescence probe 2',7'- dichlorodihydrofluorescein diacetate was utilized to detect reactive oxygen species (ROS). An in vivo mouse model was used to determine whether ALDH2 activation induces anagen. RESULTS: During the anagen phase, ALDH2 showed significantly higher intensity than that in the telogen phase, and its expression was primarily localized along the outer layer of HFs. ALDH2 activation promoted anagen phase induction by reducing ROS levels and enhancing reactive aldehyde clearance, which indicated that ALDH2 functions as a ROS scavenger within HFs. Moreover, ALDH2 activation upregulated Akt/GSK 3ß/ß-catenin signaling in HFs. CONCLUSIONS: Our findings highlight the hair growth promotion effects of ALDH2 activation in HFs and its potential as a promising therapeutic approach for promoting anagen induction.

Dissolving Candlelit Microneedle for Chronic Inflammatory Skin Diseases.

Chronic inflammatory skin diseases (CISDs) negatively impact a large number of patients. Injection of triamcinolone acetonide (TA), an anti-inflammatory steroid drug, directly into the dermis of diseased skin using needle-syringe systems is a long-established procedure for treating recalcitrant lichenified lesions of CISDs, referred to as TA intralesional injection (TAILI). However, TAILI causes severe pain, causing patients to be stressed and reluctant to undergo treatment. Furthermore, the practitioner dependency on the amount and depth of the injected TA makes it difficult to predict the prognosis. Here, candle flame ("candlelit")-shaped TA-loaded dissolving microneedles (Candlelit-DMN) are designed and fabricated out of biocompatible and biodegradable molecules. Candlelit-DMN distributes TA evenly across human skin tissue. Conjoined with the applicator, Candlelit-DMN is efficiently inserted into human skin in a standardized manner, enabling TA to be delivered within the target layer. In an in vivo skin inflammation mouse model, Candlelit-DMN inserted with the applicator effectively alleviates inflammation by suppressing inflammatory cell infiltration and cytokine gene expression, to the same extent as TAILI. This Candlelit-DMN with the applicator arouses the interest of dermatologists, who prefer it to the current TAILI procedure.

High-Dose Steroid Dissolving Microneedle for Relieving Atopic Dermatitis.

Dissolving microneedles (DMN) supplemented with therapeutic molecules have been developed to enhance transdermal delivery efficiency of topically applied drugs in a minimally invasive manner. However, the dose of the drugs in DMN system is limited owing to the low solubility of drug. In fact, although triamcinolone acetonide (TA) is one of the most widely prescribed drugs for relieving atopic dermatitis (AD), its poor dissolving nature makes it difficult to design and fabricate DMN containing therapeutic dosage of TA. In this study, TA suspension is introduced to encapsulate therapeutic dosage of TA. Sonication and composition optimization of polymers is key to fabricate high dose TA-DMN to induce particle size reduction and dispersion stability of suspension, respectively. After confirming the physical performance of TA-DMN using the selected formulation in vitro, the anti-inflammatory effects of TA-DMN are evaluated in vivo using a mouse model affected with skin inflammation to mimic AD in humans. Herein, high-dose TA-DMN is presented as a candidate agent for relieving AD and, furthermore, for wide application in the treatment of skin inflammatory diseases in which high-dose steroid drugs are required.

"Two-Cell Assemblage" Assay: A Simple in vitro Method for Screening Hair Growth-Promoting Compounds.

Alopecia arises due to inadequate hair follicle (HF) stem cell activation or proliferation, resulting in prolongation of the telogen phase of the hair cycle. Increasing therapeutic and cosmetic demand for alleviating alopecia has driven research toward the discovery or synthesis of novel compounds that can promote hair growth by inducing HF stem cell activation or proliferation and initiating the anagen phase. Although several methods for evaluating the hair growth-promoting effects of candidate compounds are being used, most of these methods are difficult to use for large scale simultaneous screening of various compounds. Herein, we introduce a simple and reliable in vitro assay for the simultaneous screening of the hair growth-promoting effects of candidate compounds on a large scale. In this study, we first established a 3D co-culture system of human dermal papilla (hDP) cells and human outer root sheath (hORS) cells in an ultra-low attachment 96-well plate, where the two cell types constituted a polar elongated structure, named "two-cell assemblage (TCA)." We observed that the long axis length of the TCA gradually increased for 5 days, maintaining biological functional integrity as reflected by the increased expression levels of hair growth-associated genes after treatment with hair growth-promoting molecules. Interestingly, the elongation of the TCA was more prominent following treatment with the hair growth-promoting molecules (which occurred in a dose-dependent manner), compared to the control group (p < 0.05). Accordingly, we set the long axis length of the TCA as an endpoint of this assay, using a micro confocal high-content imaging system to measure the length, which can provide reproducible and reliable results in an adequate timescale. The advantages of this assay are: (i) it is physiologically and practically advantageous as it uses 3D cultured two-type human cells which are easily available; (ii) it is simple as it uses length as the only endpoint; and (iii) it is a high throughput system, which screens various compounds simultaneously. In conclusion, the "TCA" assay could serve as an easy and reliable method to validate the hair growth-promoting effect of a large volume of library molecules.

Shikimic acid, a mannose bioisostere, promotes hair growth with the induction of anagen hair cycle.

Shikimic acid (SA) has recently been found to be a major component of plant stem cells. The exact effects of SA on human hair follicles (HFs) is unknown. The purpose of this study was to examine the effects of SA on hair growth. We investigated the effect of SA on an in vivo C57BL/6 mouse model. We examined the expression of mannose receptor (MR), which is a known receptor of SA, in human HFs and the effect of SA on human dermal papilla cells (hDPCs), outer root sheath cells (hORSCs), and on ex vivo human hair organ culture. SA significantly prolonged anagen hair growth in the in vivo mouse model. We confirmed expression of the MR in human HFs, and that SA increased the proliferation of hDPCs and hORSCs. It was found that SA enhanced hair shaft elongation in an ex vivo human hair organ culture. SA treatment of hDPCs led to increased c-myc, hepatocyte growth factor, keratinocyte growth factor and vascular endothelial growth factor levels and upregulation of p38 MAPK and cAMP response element-binding protein levels. Our results show that SA promotes hair growth and may serve as a new therapeutic agent in the treatment of alopecia.

Priming mobilization of hair follicle stem cells triggers permanent loss of regeneration after alkylating chemotherapy.

The maintenance of genetic integrity is critical for stem cells to ensure homeostasis and regeneration. Little is known about how adult stem cells respond to irreversible DNA damage, resulting in loss of regeneration in humans. Here, we establish a permanent regeneration loss model using cycling human hair follicles treated with alkylating agents: busulfan followed by cyclophosphamide. We uncover the underlying mechanisms by which hair follicle stem cells (HFSCs) lose their pool. In contrast to immediate destructive changes in rapidly proliferating hair matrix cells, quiescent HFSCs show unexpected massive proliferation after busulfan and then undergo large-scale apoptosis following cyclophosphamide. HFSC proliferation is activated through PI3K/Akt pathway, and depletion is driven by p53/p38-induced cell death. RNA-seq analysis shows that HFSCs experience mitotic catastrophe with G2/M checkpoint activation. Our findings indicate that priming mobilization causes stem cells to lose their resistance to DNA damage, resulting in permanent loss of regeneration after alkylating chemotherapy.

UVB-induced depletion of donor-derived dendritic cells prevents allograft rejection of immune-privileged hair follicles in humanized mice.

Dendritic cells (DCs) are key targets for immunity and tolerance induction; they present donor antigens to recipient T cells by donor- and recipient-derived pathways. Donor-derived DCs, which are critical during the acute posttransplant period, can be depleted in graft tissue by forced migration via ultraviolet B light (UVB) irradiation. Here, we investigated the tolerogenic potential of donor-derived DC depletion through in vivo and ex vivo UVB preirradiation (UV) combined with the injection of anti-CD154 antibody (Ab) into recipients in an MHC-mismatched hair follicle (HF) allograft model in humanized mice. Surprisingly, human HF allografts achieved long-term survival with newly growing pigmented hair shafts in both Ab-treated groups (Ab-only and UV plus Ab) and in the UV-only group, whereas the control mice rejected all HF allografts with no hair regrowth. Perifollicular human CD3+ T cell and MHC class II+ cell infiltration was significantly diminished in the presence of UV and/or Ab treatment. HF allografts in the UV-only group showed stable maintenance of the immune privilege in the HF epithelium without evidence of antigen-specific T cell tolerance, which is likely promoted by normal HFs in vivo. This immunomodulatory strategy targeting the donor tissue exhibited novel biological relevance for clinical allogeneic transplantation without generalized immunosuppression.

Novel effect of sildenafil on hair growth.

BACKGROUND: Sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, is known to increase the intracellular level of cyclic guanosine monophosphate (cGMP), which causes vasodilation. However, the effect of sildenafil on human hair follicles (hHFs) is unknown. OBJECTIVE: The purpose of this study was to determine the role of sildenafil in hair growth. METHODS: We investigated the expression of PDE5 in human dermal papilla cells (hDPCs) and hHFs. The effects of sildenafil on hDPC proliferation were evaluated using BrdU assays. The mRNA expression of growth factors and extracellular signal-regulated kinase (ERK) phosphorylation were investigated using real-time PCR and western blotting, respectively. Additionally, anagen induction and perifollicular vessel formation were evaluated using an in vivo mice model. RESULTS: We confirmed high expression of PDE5 in hDPCs and hHFs. Sildenafil enhances proliferation of hDPCs and up-regulates the mRNA expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), which are responsible for hair growth. Additionally, sildenafil up-regulates the levels of phosphorylated ERK and accelerates anagen induction by stimulating perifollicular vessel formation after topical application in mice. CONCLUSION: Our study demonstrates for the first time, the significant therapeutic potential of sildenafil on hair growth and its potential use in treatment of alopecia.

Minoxidil activates ß-catenin pathway in human dermal papilla cells: a possible explanation for its anagen prolongation effect.

BACKGROUND: It is believed that the length of the actively growing phase of the anagen hair cycle mainly contributes to hair length. Recent studies showed that maintenance of ß-catenin activity in the dermal papilla cells (DPCs) enables hair follicles to keep actively growing. Topical minoxidil treatment promotes hair growth in men with androgenetic alopecia, suggesting that minoxidil may prolong the actively growing phase of the anagen hair cycle. OBJECTIVE: To investigate whether minoxidil prolongs the anagen hair cycle in mice and, if so, to investigate whether minoxidil activates ß-catenin pathway in human DPCs. METHODS: Dorsal skins of C57BL/6 mice were depilated to synchronize the hair cycle. After 10 days, 3% minoxidil were topically applied daily for 10 days. Sections of back skins were stained with hematoxylin and eosin. Hair follicles were graded and hair cycle score (HCS) was calculated. Cultured human DPCs were transiently transfected with the ß-catenin responsive TCF reporter plasmid (pTopflash) and corresponding negative control reporter (pFopflash) to assess the activity of ß-catenin signaling by minoxidil. Immunofluorescence staining and immunoblot were performed to examine the expression and localization of ß-catenin in the presence or absence of minoxidil. Phosphorylation of GSK3ß, PKA and PKB were also examined by immunoblot after minoxidil treatment. RT-PCR analysis and immunoblot were employed to investigate the expression of ß-catenin pathway targets in DPCs, such as Axin2, Lef-1, and EP2. RESULTS: Modest extension of anagen phase thereby delay of catagen progression was observed by application of minoxidil in mice. Minoxidil stimulated the transcriptional activity of pTopflash but not pFopflash. Nuclear accumulation of ß-catenin was also observed after minoxidil treatment. Immunoblot further showed that minoxidil treatment increases the phosphorylation of GSK3ß, PKA and PKB. Moreover, minoxidil induced Axin2, Lef-1, and EP2 expression. CONCLUSION: Our results strongly suggest that minoxidil extends the anagen phase by activating ß-catenin activity in the DPCs.

Erythropoietin promotes hair shaft growth in cultured human hair follicles and modulates hair growth in mice.

BACKGROUND: Recent studies have shown that erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling exist in both human and mouse hair follicles (HFs). OBJECTIVE: To investigate whether dermal papilla cells (DPCs) express functional EPOR and, if so, to investigate effects of EPO on hair shaft growth in cultured human scalp hair follicles and hair growth in mice. METHODS: EPOR expression in DPCs and follicular keratinocytes was examined by RT-PCR and immunoblot. Phosphorylation of EPOR signaling pathway mediators by EPO treatment was examined by immunoblot. MTT assay was employed to check cell viability after EPO treatment. Hair shaft growth was measured in the absence or presence of EPO and matrix keratinocyte proliferation was examined by Ki-67 immunostaining in cultured hair follicles. Agarose beads containing EPO were implanted into dorsal skin of C57BL/6 mice to examine effects of EPO on hair growth in vivo. RESULTS: EPOR mRNA and protein are expressed in cultured human DPCs. EPOR signaling pathway mediators such as EPOR and Akt are phosphorylated by EPO in DPCs. EPO significantly promoted the growth of DPCs and elongated hair shafts with increased proliferation of matrix keratinocytes in cultured human hair follicles. In addition, EPO not only promoted anagen induction from telogen but also prolonged anagen phase. CONCLUSIONS: EPO may modulate hair growth by stimulating DPCs that express functional EPOR.

Identification of transcriptional targets of Wnt/beta-catenin signaling in dermal papilla cells of human scalp hair follicles: EP2 is a novel transcriptional target of Wnt3a.

BACKGROUND: Recent studies showed that Wnt signaling through the beta-catenin pathway (canonical Wnt signaling) act on mouse dermal papilla cells (DPCs) enabling hair follicles to keep growing. OBJECTIVE: To investigate whether human DPCs respond to canonical Wnt signaling and, if so, to identify target genes of Wnt/beta-catenin pathway. METHODS: Cultured human DPCs were transiently transfected with the beta-catenin responsive TCF reporter plasmid (pTopflash) and corresponding negative control reporter (pFopflash) to assess the activity of beta-catenin signaling by Wnt3a (one of the canonical Wnts). Immunofluorescence staining was also performed to localize beta-catenin in the presence or absence of Wnt3a. Microarray was carried out using Affymetrix gene chips. RT-PCR analysis and immunoblot were employed to verify microarray data. Cyclic AMP (cAMP) levels were measured using EIA assay after Wnt3a and PGE2 treatment in DPCs. RESULTS: Wnt3a significantly stimulated the transcriptional activity of pTopflash but not pFopflash. In line with this, we identified a number of genes that are regulated by Wnt3a. Some of the differently expressed genes including EP2 were confirmed by RT-PCR analysis. Immunoblot further confirmed that EP2 protein is indeed increased by Wnt3a. DPCs pretreated with Wnt3a showed higher responsiveness to PGE2 as measured by cAMP levels. CONCLUSIONS: Elucidation of the role of Wnt3a-regulated genes identified in this study including EP2 would help our understanding of hair-induction and maintenance of anagen phase.