The Effect of JAK Inhibitor on the Survival, Anagen Re-Entry, and Hair Follicle Immune Privilege Restoration in Human Dermal Papilla Cells.

Topical or systemic administration of JAK inhibitors has been shown to be a new treatment modality for severe alopecia areata (AA). Some patients show a good response to JAK inhibitors, but frequently relapse after cessation of the treatment. There have been no guidelines about the indications and use of JAK inhibitors in treating AA. The basic pathomechanism of AA and the relevant role of JAK inhibitors should support how to efficiently use JAK inhibitors. We sought to investigate the effect of JAK1/2 inhibitor on an in vitro model of AA and to examine the possible mechanisms. We used interferon gamma-pretreated human dermal papilla cells (hDPCs) as an in vitro model of AA. Ruxolitinib was administered to the hDPCs, and cell viability was assessed. The change of expression of the Wnt/ß-catenin pathway, molecules related to the JAK-STAT pathway, and growth factors in ruxolitinib-treated hDPCs was also examined by reverse transcription PCR and Western blot assay. We examined immune-privilege-related molecules by immunohistochemistry in hair-follicle culture models. Ruxolitinib did not affect the cell viability of the hDPCs. Ruxolitinib activated several molecules in the Wnt/ß-catenin signaling pathway, including Lef1 and ß-catenin, and suppressed the transcription of DKK1 in hDPCs, but not its translation. Ruxolitinib reverted IFN-γ-induced expression of caspase-1, IL-1ß, IL-15, and IL-18, and stimulated several growth factors, such as FGF7. Ruxolitinib suppressed the phosphorylation of JAK1, JAK2 and JAK3, and STAT1 and 3 compared to IFN-γ pretreated hDPCs. Ruxolitinib pretreatment showed a protective effect on IFN-γ-induced expression of MHC-class II molecules in cultured hair follicles. In conclusion, ruxolitinib modulated and reverted the interferon-induced inflammatory changes by blocking the JAK-STAT pathway in hDPCs under an AA-like environment. Ruxolitinib directly stimulated anagen-re-entry signals in hDPCs by affecting the Wnt/ß-catenin pathway and promoting growth factors in hDPCs. Ruxolitinib treatment prevented IFN-γ-induced collapse of hair-follicle immune privilege.

Effects of mesenchymal stem cell therapy on alopecia areata in cellular and hair follicle organ culture models.

Mesenchymal stem cell therapy (MSCT) has been suggested as a new therapeutic strategy for immunological disorders. There have been only a few attempts to treat alopecia areata (AA) with MSCT. MSCT efficacy and mechanism of action in treating AA are not known. We sought to investigate the effect of human hematopoietic mesenchymal stem cells (hHMSCs) on an in vitro model of AA and to explore relevant mechanisms that regulate efficacy. An AA-like environment was induced by pretreatment of human dermal papilla cells (hDPCs) with interferon gamma (IFN-γ). hHMSCs were administered to the hDPCs, and cell viability was determined. Similar studies were also conducted with human hair follicles (HFs) in culture. The change in expression of the Wnt/ß-catenin pathway and JAK/STAT pathway-related molecules and growth factors in hHMSC-treated hDPCs was also examined by reverse transcription-PCR, Western blot assay and growth factor array. Immune privilege-related molecules were examined by immunohistochemistry in HF culture models. hHMSCs enhanced the cell viability of the hDPCs. hHMSCs activated several molecules in the Wnt/ß-catenin signalling pathway, including ß-catenin and phosphorylated GSK3b, and decreased IFN-γ-induced expression of DKK1 in hDPCs. hHMSCs suppressed IFN-γ-induced expression of caspase-1, caspase-3 and IFN-γ receptor. hHMSCs induced the phosphorylation of STAT1 and STAT3 compared to controls and IFN-γ-pretreated hDPCs. hHMSC-treated HFs enhanced several growth factor mRNAs. hHMSC pretreatment modulated IFN-γ-induced expression of molecules related to HF immune privilege on HFs in organ culture. These data suggest MSCT may be a new potential therapeutic option in treating AA.

Synthesized Ceramide Induces Growth of Dermal Papilla Cells with Potential Contribution to Hair Growth.

BACKGROUND: The ceramide is known to play an important role in the formation of intracellular lipids, and play a crucial role as a barrier for skin and hair cuticle. Recent study has revealed that ceramide has potential effect on hair growth in a mouse model. However, the role of ceramide in human dermal papilla cells (hDPCs) known to play an important role in hair growth is not well understood yet. OBJECTIVE: The goal of this study was to investigate the effect of synthetic ceramides (oleyl and stearyl ceramides) on hair growth using hDPCs. METHODS: hDPCs were treated with synthesized ceramides. hDPCs viability was evaluated by MTT assay. The expression of hair growth related factors were investigated by western blot, real-time polymerase chain reaction and growth factor array. The expression of ß-catenin was confirmed by immunofluorescence. RESULTS: Treatment with ceramides increased the expression of proteins affecting cell proliferation such as Bcl-2, BAX, phosphorylated-ERK and Cyclin D1. Also, ceramides treatment were increased the expression of several growth factors, including epidermal growth factor family, and promote the expression of Wnt/ß-catenin and BMP2/4 signaling. CONCLUSION: Our data suggest that synthetic ceramides stimulates hair growth by induction proliferation of hDPCs via modulation of Wnt/ß-catenin and BMP2/4 signaling.

Prostaglandin D2-Mediated DP2 and AKT Signal Regulate the Activation of Androgen Receptors in Human Dermal Papilla Cells.

Prostaglandin D2 (PGD2) and prostaglandin D2 receptor 2 (DP2) is known to be an important factor in androgenetic alopecia (AGA). However, the effect of PGD2 in human dermal papilla cells (hDPCs) is not fully understood. The function of PGD2-induced expression of the androgen receptor (AR), DP2, and AKT (protein kinase B) signal were examined by using real time-PCR (qRT-PCR), western blot analysis, immunocytochemistry (ICC), and siRNA transfection system. PGD2 stimulated AR expression and AKT signaling through DP2. PGD2 stimulated AR related factors (transforming growth factor beta 1 (TGFß1), Creb, lymphoid enhancer binding factor 1 (LEF1), and insulin-like growth factor 1, (IGF-1)) and AKT signaling (GSK3ß and Creb) on the AR expression in hDPCs. However, these factors were down-regulated by DP2 antagonist (TM30089) and AKT inhibitor (LY294002) as well as DP2 knockdown in hDPCs decreased AR expression and AKT signaling. Finally, we confirmed that PGD2 stimulates the expression of AR related target genes, and that AKT and its downstream substrates are involved in AR expression on hDPCs. Taken together, our data suggest that PGD2 promotes AR and AKT signal via DP2 in hDPCs, thus, PGD2 and DP2 signal plays a critical role in AR expression. These findings support the additional explanation for the development of AGA involving PGD2-DP2 in hDPCs.

Various Wavelengths of Light-Emitting Diode Light Regulate the Proliferation of Human Dermal Papilla Cells and Hair Follicles via Wnt/ß-Catenin and the Extracellular Signal-Regulated Kinase Pathways.

BACKGROUND: The human dermal papilla cells (hDPCs) play an important role in regulation of hair cycling and growth. OBJECTIVE: The aim of this study was to investigate the effect of different wavelengths of light-emitting diode (LED) irradiation on the proliferation of cultured hDPCs and on the growth of human hair follicles (HFs) in vitro. METHODS: We examined the effect of LED irradiation on Wnt/ß-catenin signaling and mitogen-activated protein kinase (MAPK) pathways in hDPCs. Anagen HFs were cultured with LED irradiation and elongation of each hair shaft was measured. RESULTS: The most potent wavelength in promoting the hDPC proliferation is 660 nm and 830 nm promoted hDPC proliferation to a lesser extent than 660 nm. Various wavelengths significantly increased ß-catenin, Axin2, Wnt3a, Wnt5a and Wnt10b mRNA expression. LED irradiation significantly increased ß-catenin and cyclin D expression, and the phosphorylation of MAPK and extracellular signal-regulated kinase (ERK). HFs irradiated with 415 nm and 660 nm grew longer than control. CONCLUSION: Our result suggests that LED has a potential to stimulate hDPC proliferation via the activation of Wnt/ß-catenin signaling and ERK pathway. To our best knowledge, this is the first report which investigated that the effect of various wavelengths of LED on hDPC proliferation and the underlying mechanisms.

Repeated Microneedle Stimulation Induces Enhanced Hair Growth in a Murine Model.

BACKGROUND: Microneedle is a method that creates transdermal microchannels across the stratum corneum barrier layer of skin. No previous study showed a therapeutic effect of microneedle itself on hair growth by wounding. OBJECTIVE: The aim of this study is to investigate the effect of repeated microwound formed by microneedle on hair growth and hair growth-related genes in a murine model. METHODS: A disk microneedle roller was applied to each group of mice five times a week for three weeks. First, to identify the optimal length and cycle, microneedles of lengths of 0.15 mm, 0.25 mm, 0.5 mm, and 1 mm and cycles of 3, 6, 10, and 13 cycles were applied. Second, the effect of hair growth and hair-growth-related genes such as Wnt3a, ß-catenin, vascular endothelial growth factor (VEGF), and Wnt10b was observed using optimized microneedle. Outcomes were observed using visual inspection, real-time polymerase chain reaction, and immunohistochemistry. RESULTS: We found that the optimal length and cycle of microneedle treatment on hair growth was 0.25 mm/10 cycles and 0.5 mm/10 cycles. Repeated microneedle stimulation promoted hair growth, and it also induced the enhanced expression of Wnt3a, ß-catenin, VEGF, and Wnt10b. CONCLUSION: Our study provides evidence that microneedle stimulation can induce hair growth via activation of the Wnt/ß-catenin pathway and VEGF. Combined with the drug delivery effect, we believe that microneedle stimulation could lead to new approaches for alopecia.

A Clinicoimmunohistopathologic Study of Anetoderma: Is Protruding Type More Advanced in Stage Than Indented Type?

Background. The clinical and histopathologic classification of anetoderma are not well characterized. Objective. We aimed to investigate the clinical and histopathologic characteristics of anetoderma and to correlate clinical phenotypes with immunohistopathologic findings. Methods. We retrospectively reviewed the medical records of 30 patients with anetoderma and performed immunohistochemistry for elastin, fibrillin-1, metalloproteinase- (MMP-) 2, MMP-7, MMP-9, and MMP-12, and tissue inhibitor of metalloproteinase- (TIMP-) 1 and TIMP-2. Results. Protruding type (n = 17) had a longer disease duration and more severe loss of elastin, without changes in fibrillin, than indented type (n = 13). MMP-2 and MMP-9 showed significantly higher expressions in the dermis compared with controls (p < 0.05). MMP-7 and MMP-12 showed little expressions in both anetoderma and control tissue. TIMP-1 was highly expressed in anetoderma lesions and controls. TIMP-2 expression was variable. Conclusions. Our findings suggest that protruding type anetoderma may represent a more advanced stage and that MMP-2 and MMP-9 could be responsible for elastic fiber degradation in anetoderma.

Hair growth-promotion effects of different alternating current parameter settings are mediated by the activation of Wnt/ß-catenin and MAPK pathway.

Electrical stimulation is being used in variable skin therapeutic conditions. There have been clinical studies demonstrating the positive effect of electrical stimuli on hair regrowth. However, the underlying exact mechanism and optimal parameter settings are not clarified yet. To investigate the effects of different parameter settings of electrical stimuli on hair growth by examining changes in human dermal papilla cells (hDPCs) in vitro and by observing molecular changes in animal tissue. In vitro, cultured hDPCs were electrically stimulated with different parameter settings at alternating current (AC). Cell proliferation was measured by MTT assay. The Ki67 expression was measured by immunofluorescence. Hair growth-related gene expressions were measured by RT-PCR. In animal model, different parameter settings of AC were applied to the shaved dorsal skin of rabbit for 8 weeks. Expression of hair-related genes in the skin of rabbit was examined by RT-PCR. At low voltage power (3.5 V) and low frequency (1 or 2 MHz) with AC, in vitro proliferation of hDPCs was successfully induced. A significant increase in Wnt/ß-catenin, Ki67, p-ERK and p-AKT expressions was observed under the aforementioned settings. In animal model, hair regrowth was observed in the entire stimulated areas under individual conditions. Expression of hair-related genes in the skin significantly increased on the 6th week of treatment. There are optimal conditions for electrical stimulated hair growth, and they might be different in the cells, animals and human tissues. Electrical stimuli induce mechanisms such as the activation of Wnt/ß-catenin and MAPK pathway in hair follicles.

Mycophenolate antagonizes IFN-γ-induced catagen-like changes via ß-catenin activation in human dermal papilla cells and hair follicles.

Recently, various immunosuppressant drugs have been shown to induce hair growth in normal hair as well as in alopecia areata and androgenic alopecia; however, the responsible mechanism has not yet been fully elucidated. In this study, we investigate the influence of mycophenolate (MPA), an immunosuppressant, on the proliferation of human dermal papilla cells (hDPCs) and on the growth of human hair follicles following catagen induction with interferon (IFN)-γ. IFN-γ was found to reduce ß-catenin, an activator of hair follicle growth, and activate glycogen synthase kinase (GSK)-3ß, and enhance expression of the Wnt inhibitor DKK-1 and catagen inducer transforming growth factor (TGF)-ß2. IFN-γ inhibited expression of ALP and other dermal papillar cells (DPCs) markers such as Axin2, IGF-1, and FGF 7 and 10. MPA increased ß-catenin in IFN-γ-treated hDPCs leading to its nuclear accumulation via inhibition of GSK3ß and reduction of DKK-1. Furthermore, MPA significantly increased expression of ALP and other DPC marker genes but inhibited expression of TGF-ß2. Therefore, we demonstrate for the first time that IFN-γ induces catagen-like changes in hDPCs and in hair follicles via inhibition of Wnt/ß-catenin signaling, and that MPA stabilizes ß-catenin by inhibiting GSK3ß leading to increased ß-catenin target gene and DP signature gene expression, which may, in part, counteract IFN-γ-induced catagen in hDPCs.

Notch intracellular domain expression in various skin fibroproliferative diseases.

BACKGROUND: The effects of the Notch signaling pathway in fibroproliferative skin diseases have not been fully elucidated. OBJECTIVE: The aim of this study was to investigate the expression of activated Notch signaling molecules in various skin fibroproliferative diseases. METHODS: Immunohistochemical analysis of Notch intracellular domain (NICD) expression in keloid, hypertrophic scar, morphea, dermatofibroma, and normal control skin specimens was performed, and the clinical characteristics of patients with various skin fibroproliferative diseases were analyzed. RESULTS: NICD was highly expressed in fibroblasts of keloids and moderately to highly expressed in hypertrophic scars and dermatofibromas, whereas low or no expression was detected in the fibroblasts of normal skin specimens and morpheas. NICD was constitutively expressed in keratinocytes, endothelial cells, and immune cells in normal skin specimens. CONCLUSION: NICD was significantly expressed in human fibroproliferative skin disorders, especially keloids, suggesting that an activated Notch signaling pathway is involved in the pathogenesis of skin fibrosis.