Updated Molecular Regulation of Hair Growth and Pigmentation by Dermal Papilla Cells / 中国医学科学院学报

There is growing evidence that dermal papilla cells(DPCs)act as the organizing center to induce the cyclic hair regeneration.On one hand,DPCs secrete cytokines or growth factors to regulate the differentiation,proliferation,and migration of epithelial stem cells(EpSCs)and melanocyte stem cells(MeSCs)residing in the bulge region.On the other hand,DPCs manipulate the microenvironment(also termed as niche)for both EpSCs and MeSCs,such as the size of dermal papilla,the distance between dermal papilla and the bulge region,and the lymphatic drainage and sympathetic nerve innervation surrounding the bulge region,thereby orchestrating the cycling hair growth.Recent studies have demonstrated at least four subpopulations existing in dermal papillae,which induce the unilineage transit-amplifying epithelial cells to form the concentric multilayers of hair shafts and sheaths.In addition,emerging study has indicated that sustained psychological stress potentially leads to hyperactivation of the sympathetic nerves that innervate the bulge region.The large amount of norepinephrine released by the nerve endings forces MeSCs to rapidly and abnormally proliferate,resultantly causing the depletion of MeSC pool and the loss of hair pigment.Understanding the molecular regulation of hair growth and pigmentation by DPCs holds substantial promise for the future use of cultured DPCs

Human umbilical cord blood mesenchymal stem cells engineered to overexpress growth factors accelerate outcomes in hair growth

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are used in tissue repair and regeneration; however, the mechanisms involved are not well understood. We investigated the hair growth-promoting effects of hUCB-MSCs treatment to determine whether hUCB-MSCs enhance the promotion of hair growth. Furthermore, we attempted to identify the factors responsible for hair growth. The effects of hUCB-MSCs on hair growth were investigated in vivo, and hUCB-MSCs advanced anagen onset and hair follicle neogeneration. We found that hUCB-MSCs co-culture increased the viability and up-regulated hair induction-related proteins of human dermal papilla cells (hDPCs) in vitro. A growth factor antibody array revealed that secretory factors from hUCB-MSCs are related to hair growth. Insulin-like growth factor binding protein-1 (IGFBP-1) and vascular endothelial growth factor (VEGF) were increased in co-culture medium. Finally, we found that IGFBP-1, through the co-localization of an IGF-1 and IGFBP-1, had positive effects on cell viability; VEGF secretion; expression of alkaline phosphatase (ALP), CD133, and β-catenin; and formation of hDPCs 3D spheroids. Taken together, these data suggest that hUCB-MSCs promote hair growth via a paracrine mechanism.

Inhibitory effect of angiogenin on the expression of type Ⅰ collagen and fibronectin in dermal papilla cells from androgenetic alopecia areas / 中华皮肤科杂志

Objective To evaluate effects of angiogenin on the expression of type Ⅰ collagen and fibronectin in dermal papilla cells from androgenetic alopecia areas,and to explore its possible mechanisms.Methods Dermal papilla cells were isolated from androgenetic alopecia areas and cultured.Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of androgen receptor in dermal papilla cells of different passages,and cell counting kit-8 (CCK-8) assay to evaluate the effect of angiogenin at different concentrations of 0,10,20,40,80,160 μg/L on the proliferative activity of the dermal papilla cells cultured in a medium with or without 0.1 nmol/L dihydrotestosterone.The confluent first-passage dermal papilla cells were divided into 3 groups:control group receiving no treatment,dihydrotestosterone group treated with 0.1 nmol/L dihydrotestosterone,and dihydrotestosterone + angiogenin group treated with 0.1 nmol/L dihydrotestosterone and 80 μg/L angiogenin.After 48-hour treatment,realtime fluorescence-based quantitative PCR was conducted to measure the mRNA expression of type Ⅰ collagen gene,fibronectin and transforming growth factor-β1 (TGF-β1),and Western blot analysis to determine the protein expression of type Ⅰ collagen,fibronectin,TGF-β1,phosphorylated Smad2 (p-Smad2) and p-Smad3.Statistical analysis was done by one-way analysis of variance (ANOVA),least significant difference (LSD)-t test and t test for two independent samples.Results The mRNA expression of androgen receptor significantly decreased during the subcultivation of in vitro cultured dermal papilla cells from androgenetic alopecia areas (P ＜ 0.05).Cell proliferation assay showed that 20-160 μg/L angiogenin could evidently antagonize the inhibitory effect of 0.1 nmol/L dihydrotestosterone on the proliferation of dermal papilla cells (all P ＜ 0.05).Compared with the control group,the dihydrotestosterone group showed significantly higher mRNA expression of type Ⅰ collagen gene,fibronectin and TGF-β1.However,the mRNA expression of type Ⅰ collagen gene,fibronectin and TGF-β1 was significantly lower in the dihydrotestosterone + angiogenin group than in the dihydrotestosterone group (type Ⅰ collagen gene:1.563 ± 0.143 vs.4.329 ± 0.165;fibronectin:1.290 ± 0.063 vs.2.156 ± 0.115;TGF-β1:1.136 ± 0.098 vs.1.707 ± 0.100;all P ＜ 0.05).Moreover,angiogenin could obviously suppress the expression of type Ⅰ collagen,fibronectin,TGF-β1,p-Smad2 and p-Smad3 protein by dihydrotestosterone-induced dermal papilla cells (all P ＜ 0.05).Conclusion Angiogenin can inhibit the expression of type Ⅰ collagen and fibronectin in dermal papilla cells from androgenetic alopecia areas in vitro,which may be associated with the downregulated expression of TGF-β1 and inhibition of TGF-β1/Smad signaling pathway.

Effect of Miscanthus sinensis var. purpurascens Flower Extract on Proliferation and Molecular Regulation in Human Dermal Papilla Cells and Stressed C57BL/6 Mice / 中国结合医学杂志

<p><b>OBJECTIVES</b>To investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.</p><p><b>METHODS</b>MSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.</p><p><b>RESULTS</b>MSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).</p><p><b>CONCLUSIONS</b>The MSP flower extract may have hair growth-promotion activities.</p>

Synergistic Proliferating Effects of Electrical Stimulation and Minoxidil on Human Dermal Papilla Cells / 대한피부과학회지

BACKGROUND: Previous published clinical studies have demonstrated the positive effects of electrical stimulation (ES) on hair growth. Minoxidil (MXD) enhances hair growth by prolonging the anagen phase of hair follicles. MXD is used to promote hair growth in androgenetic alopecia. OBJECTIVE: To investigate the combined effect of ES and MXD on cultured human dermal papilla cells (hDPCs). METHODS: To investigate the combined effect of ES and MXD on cultured human dermal papilla cells (hDPCs). Methods: hDPCs were electrically stimulated with different parameter settings of alternating current. Electrically stimulated hDPCs were incubated in an MXD medium, after which cell proliferation was measured using an MTT assay. Ki-67 and β-catenin expressions were measured by immunofluorescence assay. In addition, Wnt/β-catenin pathway-related gene expressions were measured by real time-PCR, and phosphorylated ERK and AKT protein levels were measured by western blot assay. RESULTS: The combination of 8 V-1 MHz ES and MXD treatment promoted hDPC proliferation effectively, compared with that in the control, ES alone, or MXD alone treatment groups. The immunofluorescence assay showed that the expression of Ki-67 and β-catenin significantly increased in the combined treatment group. Most of the Wnt/β-catenin pathway-related gene expressions increased more with combined treatment than with the control, ES alone, or MXD alone treatments. However, there were no significant differences in the expression levels of phosphorylated ERK and AKT among the treatment groups. CONCLUSION: ES combined with MXD treatment had a synergistic effect on the proliferation of hDPCs. This might be through the synergistic activation of the Wnt/β-catenin pathway.

Herbal Extracts Induce Dermal Papilla Cell Proliferation of Human Hair Follicles

BACKGROUND: The number of people suffering from balding or hair thinning is increasing, despite the advances in various medical therapies. Therefore, it is highly important to develop new therapies to inhibit balding and increase hair proliferation. OBJECTIVE: We investigated the effects of herbal extracts commonly used for improving balding in traditional medicine to identify potential agents for hair proliferation. METHODS: The expression levels of 5alpha-reductase isoforms (type I and II) were analyzed using quantitative real-time reverse transcription polymerase chain reaction in the human follicular dermal papilla cells (DPCs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide and bromodeoxyuridine tests were used to evaluate the cell proliferation effect of herbal extracts in DPCs. The expression levels of extracellular signal-regulated kinase (ERK), Akt, cyclin D1, cyclin-dependent kinase 4 (Cdk4), B-cell lymphoma (Bcl-2) and Bcl-2-associated X protein (Bax) were measured using western blot analysis. RESULTS: The 5alpha-reductase isoform mRNAs and proteins were detected in the cultured DPCs, and the expression level of 5alpha-R2 in DPCs in the presence of the herbal extracts was gradually decreased. Herbal extracts were found to significantly increase the proliferation of human DPCs at concentrations ranging from 1.5% to 4.5%. These results show that the herbal extracts tested affected the protein expressions of ERK, Akt, cyclin D1, Cdk4, Bcl-2, and Bax in DPCs. CONCLUSION: These results suggest that herbal extracts exert positive effects on hair proliferation via ERK, Akt, cyclin D1, and Cdk4 signaling in DPCs; they also suggest that herbal extracts could be a great alternative therapy for increasing hair proliferation.

Hair Growth Promoting Potential of Phospholipids Purified from Porcine Lung Tissues

BP201, porcine lung tissue-derived phospholipids, consists of phosphatidylcholine as a major phospholipid species. BP201 promoted hair growth after application onto the shaved backs of BALB/c and C3H mice. Its effect was enhanced when applied together with minoxidil (MNX) in C3H mice. When the tissue specimens prepared from the shaved skins of BP201-treated and control mice were microscopically examined, the total numbers of hair follicles in both anagen and telogen phases of BP201-treated mice were significantly higher than those of control mice. The numbers of hair follicles in the anagen phase of BP201-treated mice were also higher than those of control mice. In combination with MNX, BP201 further increased the total number of hair follicles, but did not alter the percentage of hair follicles in the anagenic phase. BP201 also increased the proliferation of human hair follicle dermal papilla cells. Collectively, BP201 possesses hair growth promoting potential, which would suggest its use singly or in combination for hair growth products.

Arctiin blocks hydrogen peroxide-induced senescence and cell death though microRNA expression changes in human dermal papilla cells

BACKGROUND: Accumulating evidence indicates that reactive oxygen species (ROS) are an important etiological factor for the induction of dermal papilla cell senescence and hair loss, which is also known alopecia. Arctiin is an active lignin isolated from Arctium lappa and has anti-inflammation, anti-microbial, and anti-carcinogenic effects. In the present study, we found that arctiin exerts anti-oxidative effects on human hair dermal papilla cells (HHDPCs). RESULTS: To better understand the mechanism, we analyzed the level of hydrogen peroxide (H2O2)-induced cytotoxicity, cell death, ROS production and senescence after arctiin pretreatment of HHDPCs. The results showed that arctiin pretreatment significantly inhibited the H2O2-induced reduction in cell viability. Moreover, H2O2-induced sub-G1 phase accumulation and G2 cell cycle arrest were also downregulated by arctiin pretreatment. Interestingly, the increase in intracellular ROS mediated by H2O2 was drastically decreased in HHDPCs cultured in the presence of arctiin. This effect was confirmed by senescence associated-beta galactosidase (SA-ß-gal) assay results; we found that arctiin pretreatment impaired H2O2-induced senescence in HHDPCs. Using microRNA (miRNA) microarray and bioinformatic analysis, we showed that this anti-oxidative effect of arctiin in HHDPCs was related with mitogen-activated protein kinase (MAPK) and Wnt signaling pathways. CONCLUSIONS: Taken together, our data suggest that arctiin has a protective effect on ROS-induced cell dysfunction in HHDPCs and may therefore be useful for alopecia prevention and treatment strategies.

Genome-wide expression profile analysis of 3D cultured dermal papilla cells from patients with androgenic alopecia / 中华皮肤科杂志

Objective To screen genes differentially expressed between dermal papilla cells from occipital and vertex scalp of patients with androgenic alopecia (AGA) through a 3D culture system.Methods Dermal papilla cells isolated from the occipital scalp tissue of patients with AGA were cultured in a 2D system for several days.Then,the third-passage dermal papilla cells were subjected to a 3D culture with the presence of dihydrotestosterone (DHT) for 72 hours (experimental group).The dermal papilla cells isolated from the vertex scalp tissue of patients with AGA,which were cultured in a 3D system with dimethyl sulfoxide,but not DHT,served as the control group.Subsequently,total RNA was extracted from the cells and reversely transcribed into cDNA followed by labeling with Cy3 and hybridization to a NimbleGen microarray.Gene ontology (GO) and pathway analysis was carried out to screen differentially expressed genes between the experimental and control group.Real time PCR was conducted to validate the results of microarray analysis.Results As the genome-wide expression profile analysis showed,there were 622 genes differentially expressed between the experimental group and control group,of which,359 were up-regulated and 263 were down-regulated in the experimental group compared with the control group.The above results were corffirmed by real time PCR.GO analysis revealed that the up-regulated genes,such as the CHEK1 and Tobl genes,were mainly involved in the inhibition of cell proliferation and promotion of cell apoptosis,while the down-regulated genes,such as the BAMBI,EFNA3,Dlx3 and UCGC genes,were associated with the acceleration of cell proliferation as well as the growth and development of epidermis.Pathway analysis showed that cell circle-controlling molecules were the most abundant molecules.Conclusions Numerous signalling molecules and pathways are involved in the development of AGA,which are mainly responsible for the modulation of cell circle,proliferation and apoptosis.

Screening of proteins interacting with HSPC016 by yeast two-hybridization technique / 中华皮肤科杂志

Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization,HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid (named as pGBKT7-HSPC016) in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then,the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH 109. Four proteins,including forkhead family of transcription factors (FOXO 1 ), mitogen-activated protein kinase 11 (MAPK 11 ), phosphoinositide-3-kinase (PIK3R3) and liver X receptor were screened and identified. Bioinformatic analysis revealed that these proteins had close relationship with intracellular energy metabolism and translational regulation. Conclusions HSPC016 may regulate the aggregative behavior of DPCs by regulating the levels of intracellular reactive oxygen species (ROS) and interacting with signaling molecules involved in intracellular energy metabolism and translational regulation.

Promotive Effect of Minoxidil Combined with All-trans Retinoic Acid (tretinoin) on Human Hair Growth in Vitro

Minoxidil induces hair growth in male pattern baldness and prolongs the anagen phase. All-trans retinoic acid (ATRA) has been reported to act synergistically with minoxidil in vivo: they can enhance more dense hair regrowth than either compound alone. We evaluated the effect of minoxidil combined with ATRA on hair growth in vitro. The effect of co-treatment of minoxidil and ATRA on hair growth was studied in hair follicle organ culture. In cultured human dermal papilla cells (DPCs) and normal human epidermal keratinocytes, the expressions of Erk, Akt, Bcl-2, Bax, P53 and P21 were evaluated by immunoblot analysis. Minoxidil plus ATRA additively promoted hair growth in vitro, compared with minoxidil alone. In addition, minoxidil plus ATRA elevated phosphorylated Erk, phosphorylated Akt and the ratio of Bcl-2/Bax, but decreased the expressions of P53 and P21 more effectively than by minoxidil alone. Our results suggest that minoxidil plus ATRA would additively enhance hair growth by mediating dual functions: 1) the prolongation of cell survival by activating the Erk and Akt signaling pathways, and 2) the prevention of apoptosis of DPCs and epithelial cells by increasing the ratio of Bcl-2/Bax and downregulating the expressions of P53 and P21.

Effects of Calcitonin Gene-Related Peptide on the Hair Growth in Human Hair Follicle Organ Culture / 대한피부과학회지

BACKGROUND: It has recently been reported that the peripheral nervous system has an important role in the hair growth and hair cycle change, which is mediated by neuropeptides. Furthermore, the expression of various growth factors and apoptosis-related molecules are important in the hair growth and hair cycle change. Therefore, it is important that the relationship between the influence of neuropeptides and expression of various factors that regulate hair growth is analysed. OBJECTIVE: The purpose of this study was to investigate the relationship between influence of neuropeptides and expression of various hair growth-related factors in the hair follicle, after pre- treatment of cultured hair follicles and dermal papilla cells with a calcitonin gene-related peptide (CGRP). METHOD: Normal human scalp samples were obtained, and anagen hair follicles and dermal papilla cells were isolated and cultured in Dulbeco?s Modified Eagle's Medium (DMEM) with several combinations of supplements, in an air incubator with an atmosphere of 5% CO2/95%. The culture plates were divided into two groups: a control group (DMEM only) and a CGRP group(10-7M CGRP dissolved in DMEM). The results were then evaluated by measuring the linear hair growth and hair follicle morphology under a light microscope. In addition, after pre-treatment of cultured hair follicles and dermal papilla cells with CGRP, we examined changes of expression of hair growth factors (FGF-7, IGF-1, VEGF), hair growth-inhibitory factors (IL-1alpha, IL-1beta) and apoptosis-related molecules (p53, caspase-3). RESULTS: The following results were obtained: 1. The CGRP did not have statistically significant effect on the rate of linear hair growth in cultured hair follicles. However, it shortened the anagen stage of the hair cycle. 2. In hair follicles, the expression of IL-1beta, a hair growth inhibitory factor, was increased in the CGRP group, compared to the control. The expression of caspase-3, an apoptosis-related molecule, was also found to be higher than the control. In dermal papilla cells, no change in morphology, expression of hair growth factors or apoptosis-related molecules were found. However the expression of IL-1beta was increased, compared to the control. CONCLUSION: From these results, we can conclude that CGRP has a growth-inhibitory effect, especially in shortening the duration of the anagen phase without affecting the linear hair growth rate. CGRP also shows a hair growth-inhibitory effect at the molecular level in hair follicles and dermal papilla cells.

Effects of Substance P on the Expression of Various Factors to control Hair Growth in Human Hair Follicle Culture / 대한피부과학회지

BACKGROUND: Among constituents of the skin, hair follicle is an organ where nerve fibers with the most highest density are distributed. Recently, it has been reported that neuropeptides, which are secreted by nerve fibers, have important roles in the hair growth and hair cycle change, and that, the expression of various growth factors and apoptosis-related molecules are important in the hair growth and hair cycle change. Therefore, it was thought of import to analyse the relationship between the effect of neuropeptides and the expression of various factors to control hair growth in the hair follicle and hair follicle cells. OBJECTIVE: The purpose of this study was to investigate the relationship between the effect of neuropeptides and the expression of various hair growth-related factors at the level of hair follicle after pretreatment of cultured hair follicles and dermal papilla cells with SP. METHODS: Normal human scalp samples were obtained, and anagen hair follicles and dermal papilla cells were isolated and were cultured in Dulbeco's Modified Eagle's Medium (DMEM) with several combination of supplements in an atmosphere of 5% CO2/95% air incubator. We divided the culture plates into two groups; i.e. control group (DMEM only) and SP group (10-6M SP dissolved in DMEM). The results were evaluated by measuring linear hair growth and hair follicle morphology under a light microscope. Also, after pretreatment of cultured hair follicle and dermal papilla cells with SP, we examined changes of expression of hair growth factors (FGF-7, IGF-1, VEGF), hair growth-inhibitory factors (IL-1alpha, IL-1beta), and apoptosis-related molecules (p53, caspase-3). RESULTS: The following results were obtained. 1. SP did not have any statistically significant effect on the rate of linear hair growth in cultured hair follicles. However, it prolonged the anagen stage of hair cycle. 2. In hair follicles, the expression of FGF-7, a hair growth factor, was increased more than control, while the expression of caspase-3, an apoptosis-related molecule, was decreased more than control. Also, morphological changes as well as the changes of expression of hair growth factors and apoptosis-related molecules were not found in dermal papilla cells. However, the expression of IL-1beta, a hair growth-inhibitory factor, was decreased more than control. CONCLUSION: We can conclude from the results that SP has growth-stimulatory effect and especially prolongs the duration of anagen phase without affecting the rate of linear hair growth. Also, in hair follicles and dermal papilla cells, SP shows hair growth-stimulatory effect at the molecular levels.

Reconstruction and histologic characteristics of bilayer skin substitute inoculated of human hair follicle cells / 第三军医大学学报

Objective To study histologic characteristics of bilayer skin substitute reconstructed by cells from human hair follicle and whether the skin appendage can be induced based on the bilayer skin substitute.Methods After composite chitosan bilayer skin substitute was reconstructed with dermal papilla cells and outer root sheath cells or dermal sheath cells and outer root sheath cells,its histologic characteristics was investigated in vitro and after transplanted onto SD albino rats.Results Composite chitosan bilayer skin substitute reconstructed by cells from hair follicle had closely arranged epithelium cells and outstanding cornification;Epithelial cords linked with epidermis could be seen in dermis.However,there was no certain hair follicle-like structure formation either in vitro or in vivo.Conclusion Hair follicle cells are good source for skin substitute reconstruction,but it can not induce skin appendage formation through skin substitute by now.