Journal of Dermatological Science 74 (2014) 125–134
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A role of placental growth factor in hair growth Sun-Young Yoon a,b,c, Ji-Seon Yoon a,b,c, Seong Jin Jo a,b,c, Chang Yup Shin a,b,c, Jong-Yeon Shin d,e, Jong-Il Kim d,e, Ohsang Kwon a,b,c,*, Kyu Han Kim a,b,c,** a
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Institute of Human-Environment Interface Biology, Seoul National University Medical Research Center, Seoul, Republic of Korea c Laboratory of Cutaneous Aging and Hair Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea d Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, Republic of Korea e Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2013 Received in revised form 24 January 2014 Accepted 24 January 2014
Background: The dermal papilla (DP) comprises specialized mesenchymal cells at the bottom of the hair follicle and plays a pivotal role in hair formation, anagen induction and the hair cycle. In this study, DPs were isolated from human hair follicles and serially subcultured. From each subculture at passages 1, 3, and 5 (n = 4), we compared gene expression profiles using mRNA sequencing. Among the growth factors that were down-regulated in later passages of human DP cells (hDPCs), placental growth factor (PlGF) was selected. Objective: To elucidate the effect of PlGF on hair growth. Methods: We evaluated the effect of PlGF on hDPCs and on ex vivo hair organ culture. We investigated the effect of PlGF on an in vivo model of depilation-induced hair regeneration. Results: We confirmed that the mRNA and protein expression levels of PlGF significantly decreased following subculture of the cells. It was shown that PlGF enhanced hair shaft elongation in ex vivo hair organ culture. Furthermore, PlGF significantly accelerated hair follicle growth and markedly prolonged anagen hair growth in an in vivo model of depilation-induced hair regeneration. PlGF prevented cell death by increasing the levels of phosphorylated extracellular signal-regulated kinase (ERK) and cyclin D1 and promoted survival by up-regulation of phosphorylated Akt and Bcl2, as determined by Western blotting. Conclusion: Our results suggest that PlGF plays a role in the promotion of hair growth and therefore may serve as an additional therapeutic target for the treatment of alopecia. ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Dermal papilla Gene expression profiles PlGF Hair growth
1. Introduction The hair follicle (HF) is a complicated organ, composed of multiple layers of epithelial cells and a mass of mesenchymal cells called the dermal papilla (DP) [1]. The DP is a mesenchyme-derived structure located at the bottom of the HF. The DP carries nutrients to enhance new hair growth, and contains receptors for androgens and other diverse hormones [2]. Moreover, the DP is necessary for the induction and maintenance of follicular development, mediates the hair cycle, and is thought to supply inductive signals
* Corresponding author at: Department of Dermatology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 1996; fax: +82 2 742 7344. ** Corresponding author at: Department of Dermatology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 2410; fax: +82 2 742 7344. E-mail addresses: [email protected] (O. Kwon), [email protected] (K.H. Kim).
required for hair outgrowth [3,4]. It is well known that abnormalities in the functions of the DP are the main causes of imbalance in follicle growth cycling and hair loss. Furthermore, the DP number dictates the size and shape of the hair, and degeneration of the DP population in mice leads to different types of hair thinning and loss [5]. However, cultured DP cells (DPCs) have been shown to exhibit significant loss of their distinct characteristics following subculture [6]. Thus, studies of specific genes related to the function of DP in the HF are necessary to further our knowledge of human hair growth. In this study, placental growth factor (PlGF) was selected as a target gene that was down-regulated in later passages of human DPCs (hDPCs). Anagen development is associated with angiogenesis, and inhibition of angiogenesis leads to suppression of anagen development [7–9]. PlGF is a member of the vascular endothelial growth factor (VEGF) subfamily and binds to VEGF receptor-1 (Flt-1), or neuropilin-1, which functions in survival, proliferation, and migration in endothelial cells, smooth muscle cells, and
0923-1811/$36.00 ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2014.01.011
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hematopoietic myeloid cells [10,11]. In adult skin, PlGF expression is up-regulated in association with both physiological and pathological neoangiogenesis, such as hair follicle cycles, wound healing, and ischemia [7,10,12–14]. In particular, PlGF is expressed in outer root sheath keratinocytes during the anagen phase of the hair follicle cycle, whereas expression of PlGF is not observed during the catagen and telogen phases [15]. While studies have demonstrated that PlGF increases cutaneous vascularization and vascular permeability, the effects of PlGF on human hair growth have not been investigated. In this study, we found that PlGF enhanced hair shaft elongation in ex vivo hair organ culture, significantly accelerated hair follicle growth, and markedly prolonged anagen hair growth in an in vivo model of depilation-induced hair regeneration. Our results demonstrated that PlGF could stimulate hair growth in both in vitro and in vivo models. 2. Materials and methods 2.1. Ethics statement This study was approved by the Institutional Review Board at the Seoul National University Hospital (approval number C-1206006-412), and all subjects provided written informed consents. All experimental procedures using human materials were conducted according to the principles described in the Declaration of Helsinki. 2.2. Isolation of human HFs and DP Skin biopsy specimens were obtained from the occipital scalp region of healthy volunteers who had not received any medication for at least 1 month. HFs were isolated under a stereo dissecting microscope, and DPs were obtained from individually isolated HFs as previously described [16,17]. HFs that were morphologically considered to be in the anagen stage were used in this study. 2.3. Cell culture The methods used for isolating and culturing hDPCs have been described previously [16]. hDPCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Welgene), 10 ng/mL of basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), and 1 antibiotic/antimycotic solution (Gibco BRL, Gaithersburg, MD, USA), which included penicillin and streptomycin. Cells were incubated at 37 8C in a 5% CO2 incubator. 2.4. mRNA sequencing All samples were isolated from human HF tissues of different individuals. hDPs were isolated from human HFs and were serially subcultured. From each subculture at passages 1, 3, and 5 (n = 4), total RNA from hDPCs was isolated using an RNeasy Mini kit (Qiagen, GmbH, Hilden, Germany). From the mRNA-Seq sample preparation, sequencing libraries were generated according to the standard protocol of Illumina Inc. for high-throughput sequencing. We hybridized 10 pM of each library to a flow cell, with a single lane for each sample, and used an Illumina cluster station for cluster generation. Finally we generated 50-bp paired end sequences using the Illumina GAIIx sequencer (Illumina Inc., San Diego, CA, USA). We used the standard Illumina pipeline (Cassava 1.6) with default options to analyze the images and extract base calls in order to generate fastq files. The RNA sequencing reads were aligned to the NCBI human reference genome assembly (NCBI Build 36.3) using GSNAP [18] with an allowance for 5% mismatches. The expression levels for 36,742 reference sequence
(RefSeq) genes were measured by using uniquely aligned RNA sequencing reads. For each gene, the number of reads aligned to it (raw read count) was normalized by reads per kilobase per million mapped reads (RPKM) [19]. 2.5. Human HF organ culture Human scalp HFs were isolated and cultured as described previously [20,21]. Each dissected HF was cut from the bottom of the DP into a piece with a length of approximately 3.5 mm and was cultured in Williams’ E medium (Gibco BRL) supplemented with 10 ng/mL hydrocortisone, 10 mg/mL insulin, 2 mM L-glutamine, and 1 antibiotic/antimycotic solution (Gibco BRL) containing penicillin and streptomycin. Follicles were maintained at 37 8C in a 5% CO2 atmosphere. hHFs were treated with 10 or 50 ng/mL PlGF (R&D Systems, Minneapolis, MN, USA) and 1 mM minoxidil (Sigma, St Louis, MO, USA, MNX, positive control) for 8 days (n = 3). In all experiments, the tissue culture medium was changed every other day, and HF elongation was measured directly at 4 and 8 days of culture using a stereo microscope (Olympus, Tokyo, Japan). A total of 180 HFs from 3 different volunteers (60 follicles per volunteer) were analyzed in each growth condition. To evaluate the effect of anti-Flt1 neutralizing antibody, hHFs were treated with 500 or 1000 ng/mL anti-Flt1 neutralizing antibody and 1 mM MNX for 6 days (n = 4). In this experiment, the tissue culture medium was changed every third day, and HF elongation was measured directly at 3 and 6 days of culture. A total of 240 HFs from 4 different volunteers (60 follicles per volunteer) were analyzed in each growth condition. 2.6. Immunofluorescence staining Immunofluorescence staining was performed on 5-mm frozen sections of hHFs as previously described [21]. The following antibodies were used: anti-PlGF (Abcam, Cambridge, MA, USA), anti-Flt1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiKi67 (DAKO, Carpinteria, CA, USA), and anti-CD11b (BD Pharmingen, San Diego, CA, USA). Immunofluorescence images were acquired using Zeiss LSM 510 META confocal microscopy software. Confocal parameters for the pinhole, optical slide, detector gain, amplifier offset, amplifier gain, contrast, and brightness were kept constant between samples. For quantitative analyses, the number of Ki-67-positive cells was counted and normalized to the number of 40 ,6-diamidion-2-phenylindole (DAPI)-stained cells using 3dimensional software (Imaris Version 6.1, Bitplane AG, Switzerland). The number of CD11b-positive cells was counted using LAS AF Lite software (Leica Microsystems, Germany). Immunofluorescence staining for blood vessels was performed on 10-mm-thick frozen sections using anti-CD31antibody (BioLegend, San Diego, CA, USA). CD31-stained areas were measured using the Image J program (by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) as described previously [22]. To analyze apoptotic cells, we performed TUNEL labeling (In Situ Cell Death Detection Kit; Fluorescein, Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (blue fluorescence). Immunofluorescence images were obtained using the Leica Application Suite Advanced Fluorescence Software (LAS AF, Leica Microsystems, Heerbrugg, Switzerland). 2.7. Quantitative real time-polymerase chain reaction Total RNA was isolated from hDPCs using RNA iso Plus (Takara Bio Inc., Otsu, Shiga, Japan) and treated with DNase I (Roche Pharmaceuticals, Welwyn Garden City, UK) to remove genomic DNA. We used 1 mg of total RNA for the cDNA synthesis reaction,
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which was performed using a First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. To quantitatively estimate mRNA expression, polymerase chain reaction (PCR) was performed on a 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (Takara Bio Inc.) according to the manufacturer’s instructions. Primer information for human and mouse genes is given in Supplementary Tables S1 and S2. All experiments with SYBR Green were performed in triplicate and were independently repeated more than 3 times.
epidermis to the subcutaneous fat. Bulb diameter was measured in H&E-stained sections at the level of the largest diameter (‘‘Auber’s line’’) of the hair bulbs with clearly visible DP (n = 80 HFs/mouse group) [8]. For quantitative analyses, the Image J program (National Institutes of Health (NIH), Bethesda, MD, USA) was used. All animal experimental procedures were carried out at the animal facility of the Seoul National University Hospital in accordance with national and institutional guidelines.
2.8. Western blot analysis
The methods used for the anagen induction assay have been described previously [25]. The back skin of 8-week-old C57BL/6 female mice in the telogen phase was shaved with a clipper. Vehicle (phosphate-buffered saline [PBS] containing 0.1% bovine serum albumin [BSA], n = 3), PlGF (300 ng in 30 mL PBS containing 0.1% BSA, n = 4), or MNX (60 mg in 30 mL PBS containing 0.1% BSA, n = 4) were applied via intradermal injection twice every day. At 41 days, skin samples were obtained at the site of intradermal injection for histological analysis of hematoxylin and eosin (H&E)stained paraffin sections. Anagen induction scores were calculated by using assigned score values (telogen = 1, anagen I–VI = 2–7) [26–28]. The resulting values were added and divided by the number of HFs (n = 70 HFs/mouse group). In the histological analysis by H&E staining, skin thickness was measured as the distance from the epidermis to the subcutaneous fat by using the Image J program (NIH, Bethesda, MD, USA).
Total protein from hDPCs was extracted using RIPA lysis buffer (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Amersham, Buckinghamshire, UK) using a wet transfer system. The blotted membranes were incubated with the respective primary antibodies at 4 8C. The following antibodies were used: anti-total ERK, antiphosphorylated ERK, anti-total Akt, anti-phosphorylated Akt, anti-Bcl2, anti-Bax, anti-cyclin D1 (Cell Signaling Technology, Beverly, MA, USA), p53, anti-b-actin (Santa Cruz Biotechnology) and Flt1 (R&D Systems, Minneapolis, MN, USA). Membranes were probed with an anti-mouse, anti-rabbit-, or anti-goat-IgGhorseradish peroxidase conjugates (Santa Cruz Biotechnology) for 1 h at room temperature. Antibody–antigen complexes were detected using the ECL system (Amersham Pharmacia Biotech, Little Chalfont, UK). 2.9. RNA interference Knockdown of PlGF was carried out with RNA interference using small interfering RNAs (siRNAs). Control scrambled siRNA was used as a negative control siRNA (Bioneer, Daejeon, Korea). PlGF siRNA (siRNA No. 1115569) was purchased from Bioneer. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Whenever cells were transfected with PlGF siRNA, efficiency of PlGF siRNA was measured by quantitative real-time PCR. Compared with control scrambled siRNA, PlGF siRNA indicated approximately 90% efficiency. 2.10. BrdU-ELISA For the quantification of cell proliferation, a BrdU-ELISA (Roche, Mannheim, Germany) was used according to the manufacturer’s instructions. hDPCs were incubated with 10 or 50 ng/mL PlGF. After 48 or 72 h, incorporated BrdU levels were measured by ELISA and expressed as the absorbance of the treated samples relative to that of the untreated control.
2.12. Anagen hair induction test in C57BL/6 mice
2.13. Statistical analysis Statistical significance was determined using Student’s t-test. Pvalues were 2-tailed, and significance was accepted at P < 0.05. In the case of organ culture, a paired Student’s t-test was used for statistical analysis. 3. Results 3.1. mRNA and protein expression levels of PlGF significantly decreased following subculture of hDPCs DPs were isolated from human HFs and were serially subcultured. In each subculture at passages 1, 3, and 5 (n = 4), we compared the gene expression profiles of the cells using mRNA sequencing. From this gene expression analysis, we found that 232 genes were up-regulated (P < 0.01) and 2249 genes were downregulated (P < 0.01) in later passages of hDPCs. The RNA sequencing data were uploaded in EBI-SRA under accession number PRJEB5104. Among the down-regulated growth factors, PlGF was selected. We found that mRNA and protein expression levels of PlGF significantly decreased in later passages of cells (Fig. 1A and B). In addition, we also confirmed that PlGF secretion was reduced following subculture of hDPCs, as determined using enzyme-linked immunosorbent assay (ELISA; Fig. 1C).
2.11. In vivo model of depilation-induced hair regeneration 3.2. PlGF and Flt1 were expressed in hDPCs and hHFs The back skin of 8-week-old C57BL/6 female mice in the telogen phase was depilated using wax as described previously [23], resulting in synchronized induction of new anagen follicle growth. Vehicle (phosphate-buffered saline [PBS] containing 0.1% bovine serum albumin [BSA], n = 5), PlGF (300 ng in 30 mL PBS containing 0.1% BSA, n = 5), or MNX (60 mg in 30 mL PBS containing 0.1% BSA, n = 5) was applied via intradermal injection twice a day and skin samples were obtained at days 5 and 21 for histological analysis. The length of the hair follicles was measured as the distance from the bottom of the hair bulbs to the epidermis (n = 50 HFs/mouse group) [24]. Skin thickness was measured as the distance from the
We confirmed the expression of PlGF and Flt1, a PlGF receptor, by immunofluorescence staining. In hHFs, in particular, PlGF was highly expressed in the DP, and Flt1 was highly expressed in hair matrix keratinocytes (Fig. 2A and B). We also found that PlGF and Flt1 were expressed in hDPCs (Supplementary Fig. S1A and B). In addition, PlGF was expressed in human outer root sheath cells (hORSCs), which are keratinocytes of human scalp HFs (Supplementary Fig. S1C). We also observed that PlGF and Flt1 were not expressed during catagen phage of human hair follicle (Supplementary Fig. S2).
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Fig. 1. PlGF mRNA and protein levels significantly decreased in later passages of hDPCs. DPs were isolated from human HFs and subcultured (passages 1, 3, and 5). (A) Total RNA from hDPCs was isolated, and mRNA expression levels were measured by quantitative real-time PCR (q-PCR). (B) hDPCs were lysed and analyzed by Western blotting. (C) Supernatants of the different passages of hDPCs were collected, and the amount of PlGF secretion was measured by ELISA. Results are expressed as the mean standard error (SE). **P < 0.01, *P < 0.05 versus passage 1 (q-PCR and Western blot) or 3 (ELISA).
3.3. PlGF enhanced hair shaft elongation in ex vivo hair organ culture hHFs were treated with 10 or 50 ng/mL PlGF or 1 mM minoxidil (MNX), a positive control, for 8 days. We found that treatment with 50 ng/mL PlGF significantly enhanced hair shaft elongation after 8 days, as compared with that for the control group. In addition, treatment with 1 mM MNX also markedly increased hair growth after 8 days, as compared with that for the control group (Fig. 3A and B). We performed immunofluorescence staining for Ki-67, a proliferation marker, after HFs were treated with PlGF or MNX for 8 days. For quantitative analyses, the number of Ki67-positive (red fluorescence) cells was counted and normalized to the number of DAPI-stained cells (40 ,6-diamidion-2-phenylindole, blue fluorescence) in follicular matrix keratinocytes in the hair bulb. Compared with the control group, treatment with PlGF or MNX significantly increased the number of Ki-67-positive keratinocytes (Fig. 3C, P < 0.05). These results suggested that PlGF induced proliferation in hair matrix keratinocytes. In addition, treatment with an antiFlt-1 neutralizing antibody significantly reduced hair shaft elongation, as compared with that in the control group (Fig. 3D and E). These results suggested that PlGF exerted a direct effect on hair growth by binding Flt-1. 3.4. PlGF accelerated hair follicle growth and prolonged anagen hair growth We investigated whether PlGF would accelerate hair re-growth and prolong the anagen phase after depilation-induced hair regeneration. In histological analysis, measurement of hair follicle length, skin thickness, and bulb diameter is a well-known method to classify the hair cycle stages [8,24]. At day 5, the length of hair follicles was significantly increased in PlGF- and MNX-treated mice
compared with vehicle-treated mice (Fig. 4A and B, P < 0.01). The back skin of PlGF- or MNX-treated mice was thicker than that of vehicle-treated mice (Fig. 4C). These results suggested that PlGF significantly accelerated hair follicle growth and induce faster anagen entry compared with the vehicle-treated group. It is well known that catagen development induces apoptosis in hair matrix keratinocytes and leads to reduce the diameter of the hair bulb [27,29]. At day 21, hair bulbs in PlGF- and MNX-treated mice were thicker than those in vehicle-treated mice (Fig. 4D and E, P < 0.01). At day 21, to evaluate apoptotic cells, immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. Compared with the control group, the PlGF- and MNX-treated groups showed a decreased number of TUNEL-positive cells (apoptosis, green fluorescence, Fig. 4F) in keratinocytes of the hair bulb. Therefore, these results suggested that PlGF markedly prolonged anagen hair growth, associated with increased thickness of hair bulbs and reduced apoptosis of hair matrix keratinocytes. To investigate whether PlGF promoted angiogenesis, immunofluorescence staining for CD31, a blood vessel marker, was performed. Compared with the vehicletreated group, the PlGF-treated group exhibited an increase in the average area of vessels (green fluorescence, Fig. 4G). PlGF has been reported to act as a strong chemoattractant for monocytes/ macrophages [30], and macrophages possess the ability to induce hair growth [31]. At day 21, to examine whether PlGF induced the recruitment of macrophages, we performed immunofluorescence staining for CD11b, a macrophage marker. Compared with the control group, the PlGF-treated group exhibited increased numbers of CD11b-positive cells (green fluorescence, Fig. 4H). Furthermore, we performed an anagen induction test, a method to evaluate the effect of PlGF on hair cycling. We could also observe skin pigmentation at the site of intradermal injection in PlGF- and
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Fig. 2. Expression of PlGF and Flt1. Immunofluorescent staining of PlGF (A, red fluorescence) and Flt1 (B, red fluorescence) was performed in hHFs. Nuclei were stained by DAPI (blue fluorescence). Original magnifications: 400, 1000.
MNX-treated group (Fig. 5A). Anagen induction score and skin thickness increased in PlGF- and MNX-treated group compared with the control group (Fig. 5B–D). These results suggested that PlGF treatment induced anagen hair cycle in telogen mouse skin. 3.5. PlGF promoted the survival of hDPCs Next, we performed Western blotting to analyze the expression of proteins related to the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) and phosphotidylinositol 3-kinase (PI3K)/Akt pathways, which play key roles in multiple cellular processes, such as apoptosis, cell proliferation, cell survival, transcription and cell migration [11]. We found that PlGF treatment markedly increased the phosphorylation of Akt and ERK and the expression of Bcl2 and cyclin D1 in a time- and dosedependent manner (Fig. 6A and B). Inhibition of Akt or ERK suppressed the mRNA expression of Flt1 and PlGF (Fig. 6C and D). These results suggested that PlGF prevented cell death by increasing the phosphorylation of ERK and the expression of cyclinD1 and promoted cell survival by up-regulating Akt phosphorylation and Bcl2 expression. Next, to elucidate the biological function of PlGF, hDPCs were transfected with PlGF siRNA for 24 or 48 h and analyzed by Western blotting. We found that suppression of PlGF decreased the phosphorylation of ERK and expression of cyclin D1. The phosphorylation of Akt was also significantly decreased (Supplementary Fig. S3A and B). In addition, suppression of PlGF dramatically increased the expression of Bax and p53, which are known to promote apoptosis (Supplementary Fig. S3A and B). Moreover, after transfection with PlGF siRNA for 48 h, we confirmed that PlGF knockdown markedly reduced the mRNA expression of cyclin D1 (Supplementary Fig. S3C). These data suggested that PlGF knockdown inhibited
proliferation through suppression of ERK and cyclin D1 and prevented survival by downregulation of Akt phosphorylation and upregulation of p53 and Bax expression. Furthermore, we also found that PlGF treatment increased the number of hDPCs and enhanced the proliferation of hDPCs, as determined by BrdU-ELISA (Supplementary Fig. S4). 3.6. PlGF enhanced the mRNA expression of some representative genes related to hair induction or proliferation of hDPCs Next, we determined the mRNA expression levels of some representative genes related to hair induction or proliferation of hDPCs. After treatment with PlGF for 30 min, mRNA expression levels of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and VEGF, which are known to stimulate hair growth, were markedly increased (P < 0.05), whereas the expression of transforming growth factor beta 1 (TGF-b1), an inhibitor of hair growth, was not changed (Fig. 6E). After treatment with PlGF for 24 h, the mRNA expression levels of versican and alkaline phosphatase (ALP), which maintain the inductive properties of the DP, significantly increased (Fig. 6F and G). In addition, mRNA expression levels of cyclin D1 and cyclin E1, markers of proliferation, increased (Fig. 6F and G). Thus, these results suggested that PlGF enhanced the expression of some representative genes related to hair induction or proliferation of hDPCs. To further support the role of PlGF in hDPCs, hDPCs were transfected with PlGF siRNA. Suppression of PlGF markedly reduced the mRNA expression levels of PlGF receptors, e.g., Flt1 and neuropilin-1, and significantly decreased the expression levels of IGF-1, HGF, fibroblast growth factor 2 (FGF-2), cyclooxygenase 2 (COX-2), and c-myc, which stimulate hair growth (Supplementary Fig. S5, P < 0.01).
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Fig. 3. PlGF enhanced hair shaft elongation in ex vivo hair organ culture. (A and B) hHFs were treated with 10 or 50 ng/mL PlGF and 1 mM minoxidil (MNX, positive control) for 8 days (n = 3). HF elongation was measured directly at 4 and 8 days of culture using a stereo microscope. (C) 5-mm frozen sections of hHFs from 3 different individuals were analyzed for proliferation (Ki67-positive, red fluorescence) in keratinocytes of the hair bulb. Nuclei were counterstained with DAPI (blue fluorescence). For quantitative analyses, the number of Ki67-positive cells was counted and normalized to the number of DAPI-stained cells. (D and E) hHFs were treated with 500 or 1000 ng/mL anti-Flt1 neutralizing antibody and 1 mM MNX for 6 days (n = 4). HF elongation was measured directly at 3 and 6 days of culture using a stereo microscope. Results are expressed as the mean SE. *P < 0.05 versus the control. Numbers next to the lines are percentage ratios compared to the control. Scale bar = 100 mm; original magnification, 200.
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Fig. 4. PlGF accelerated hair follicle growth and prolonged the anagen hair cycle. (A and D) At day 5 (A) or day 21 (D), skin samples were obtained for histological analysis of H&E-stained paraffin sections. (B and C) At day 5, the length of the hair follicles and skin thickness were measured in H&E-stained sections. (E) Bulb diameter was measured at the level of the largest diameter (‘‘Auber’s line’’) of hair bulbs. (F–H) At day 21, immunofluorescent staining for TUNEL (F, apoptosis marker, green fluorescence), CD31 (G, blood vessel marker, green fluorescence) and CD11b (H, macrophage marker, green fluorescence) was performed. Numbers on the bars are percent ratios compared to the control. Results are expressed as the mean SE. **P < 0.01 versus the control group.
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Fig. 5. PlGF induced anagen hair cycle in telogen mouse skin. (A) Hair growth was restricted to the site of intradermal injection in PlGF- and MNX-treated C57BL/6 mice. (B) Skin samples were obtained for histological analysis of H&E-stained paraffin sections. (C) Anagen induction scores were calculated and assigned score values (telogen = 1, anagen I–VI = 2–7). (D) Skin thickness was measured as the distance from the epidermis to the subcutaneous fat. Numbers on the bars are relative percentages compared to control. Results are expressed as the mean SE. **P < 0.01, *P < 0.05 versus the control group. Scale = 100 mm; Original magnification, 200.
4. Discussion In this study, PlGF was selected as one of the genes that were down-regulated in later passages of hDPCs. Hair matrix keratinocytes are known to be important for hair growth, and the DP regulates the activity of matrix keratinocytes [6]. We confirmed that PlGF and Flt1, a PlGF receptor, were expressed in hDPCs and HFs. Furthermore, we found that PlGF increased the proliferation of human hair matrix keratinocytes, as determined by immunofluorescence staining for Ki-67, a proliferation marker. Interestingly, in this study, we found that PlGF enhanced hair shaft elongation in ex vivo hair organ culture, whereas hair growth was inhibited by blockade of Flt-1, a PlGF receptor. These results suggested the possibility that PlGF, a paracrine factor secreted from DPCs, acted on neighboring follicular matrix keratinocytes and had a direct stimulatory effect on hair growth by binding Flt-1. It has been reported that PlGF is expressed in ORS during the angiogenic phase of the hair follicle cycle [7,15] and we also observed that PlGF was expressed in hORSCs, which are keratinocytes of human scalp hair follicle. To investigate a paracrine effect of PlGF a little more deeply, further studies need to be undertaken to explore the effect of PlGF on hORSCs, which are keratinocytes of hHFs. Some reports have indicated that PlGF-deficient mice or PlGFtransgenic mice was associated with vascularization and vessel permeability but did not show a certain noticeable changes in hair phenotypes [12,32,33]. Otherwise, in our study, in an in vivo model of depilation-induced hair regeneration, PlGF significantly accelerated hair follicle growth, and markedly prolonged anagen hair growth. Furthermore, through an anagen hair induction test in C57BL/6, we could confirm that PlGF treatment induced anagen hair growth in telogen mouse skin due to the promoting effect on the hair cycling. Overexpression of PlGF strongly increases vascularization and enhances vessel permeability [33,34]. Additionally, perifollicular angiogenesis has been shown to be correlated with up-regulation of VEGF expression in follicular keratinocytes of the outer root sheath during the anagen phase [8]. Transgenic overexpression of VEGF improves perifollicular vascularization and accelerates murine hair regrowth after depilation. In the current study, we
showed that PlGF could increase the average area of vessels. Thus, this increase in angiogenesis by PlGF may play a role in improving hair growth. In this study, we also found that PlGF induced the recruitment of macrophages. PlGF is a strong chemoattractant for monocytes/ macrophages [30], and the increased expression of PlGF in K14PlGF diabetic wounds correlates with enhanced monocyte/ macrophage recruitment at the wound site [12]. According to Osaka et al., macrophages possess the ability to induce hair growth [31]. Therefore, their recruitment by PlGF is suggestive of its role as a chemotactic factor and it may have an indirect stimulatory effect on hair growth via macrophage. In human keratinocytes, mRNA and protein expression of PlGF were increased by epidermal growth factor (EGF), TGFa, and TGFb, key cytokines in wound healing, as well as interleukin 6 (IL-6), an inflammatory cytokine [13]. mRNA levels of PDGF, FGF-2 and VEGF, which play roles in wound healing, have been reported to show a significant increase in wounds of diabetic mice, following transfection with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) [12]. Our study also demonstrated that in hDPCs, PlGF treatment significantly increased mRNA expression of IGF-1, HGF, and VEGF, which are thought to stimulate hair growth, whereas the mRNA level of TGF-b1, an inhibitor of hair growth, was not changed. Some reports have indicated that PlGF promoted the survival function of endothelial cells via induction of the expression of the anti-apoptotic gene, survivin [35]. Moreover, in human dermal microvascular endothelial cells, survival was enhanced by induction of Bcl-2 expression [36]. In Western blot analysis, we observed that in hDPCs, PlGF increased the expression of Bcl-2, which functions in survival. Furthermore, we also found that PlGF clearly increased the protein expression of cyclin D1 and the phosphorylation of ERK and Akt, which are known to promote survival and prevent cell death. In addition, PlGF treatment increased the number of hDPCs and enhanced the proliferation of DPCs, as determined by Brdu-ELISA. Taken together, these results showed that PlGF secreted from hDPCs may exhibit an autocrine function on hDPCs. In summary, our results demonstrated that PlGF markedly stimulated hair growth in both in vitro and in vivo models. We
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Fig. 6. PlGF promoted the survival of hDPCs. (A and B) hDPCs were incubated with the indicated concentrations of PlGF. After 24 (A) or 72 h (B), cells were lysed and analyzed by Western blotting. (C and D) hDPCs were treated with 1 mM LY294002 (an inhibitor of Akt, C) or PD98059 (an inhibitor of ERK, D) for 48 (C) or 24 h (D). Total RNA was isolated and mRNA expression was analyzed by quantitative real-time PCR. (E and F) Cells were treated with 50 ng/mL PlGF for 30 min (E) or 24 h (F), total RNA was isolated, and mRNA expression was analyzed by quantitative real-time PCR. (G) hDPCs were treated with the indicated concentrations of PlGF for 24 h, total RNA was isolated, and mRNA expression was analyzed by RT-PCR. Results are expressed as the mean SE. *P < 0.05 versus the control. ALP, alkaline phosphatase.
propose that PlGF plays an important role as an inducer of hair follicle growth and, therefore, may serve as an additional therapeutic target for the treatment of alopecia. Funding sources This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No.: A103017); by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (No.: 2012R1A1A2005302); partially by a research agreement with AmorePacific Corporation, Republic of Korea.
Acknowledgements We thank Elizabeth Park, Boston University School of Medicine for her assistance during the manuscript preparation. This study was presented at the 7th World Congress for Hair Research in Edinburgh, Scotland, UK, in May 2013. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.01.011.
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[20] Philpott MP, Green MR, Kealey T. Human hair growth in vitro. J Cell Sci 1990;97:463–71. [21] Yoon SY, Kim KT, Jo SJ, Cho AR, Jeon SI, Choi HD, et al. Induction of hair growth by insulin-like growth factor-1 in 1763 MHz radiofrequency-irradiated hair follicle cells. PLoS ONE 2011;6:e28474. [22] Lee BL, Kim WH, Jung J, Cho SJ, Park J-W, Kim J, et al. A hypoxia-independent up-regulation of hypoxia-inducible factor-1 by AKT contributes to angiogenesis in human gastric cancer. Carcinogenesis 2008;29:44–51. [23] Paik SH, Yoon JS, Ryu HH, Lee JY, Shin CY, Min KH, et al. Pretreatment of epidermal growth factor promotes primary hair recovery via the dystrophic anagen pathway after chemotherapy-induced alopecia. Exp Dermatol 2013;22:496–9. [24] Yano K, Brown LF, Lawler J, Miyakawa T, Detmar M. Thrombospondin-1 plays a critical role in the induction of hair follicle involution and vascular regression during the catagen phase. J Invest Dermatol 2003;120:14–9. [25] Paus R, Stenn KS, Link RE. The induction of anagen hair growth in telogen mouse skin by cyclosporine A administration. Lab Invest 1989;60:365–9. [26] Arck PC, Handjiski B, Peters EMJ, Hagen E, Klapp BF, Paus R. Topical minoxidil counteracts stress-induced hair growth inhibition in mice. Exp Dermatol 2003;12:580–90. [27] Mu¨ller-Ro¨ver S, Handjiski B, van der Veen C, Eichmu¨ller S, Foitzik K, McKay IA, et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J Invest Dermatol 2001;117:3–15. [28] Maurer M, Peters EM, Botchkarev VA, Paus R. Intact hair follicle innervation is not essential for anagen induction and development. Arch Dermatol Res 1998;290:574–8. [29] Foitzik K, Lindner G, Mueller-Roever S, Maurer M, Botchkareva N, Botchkarev V, et al. Control of murine hair follicle regression (catagen) by TGF-b1 in vivo. FASEB J 2000;14:752–60. [30] Clauss M, Weich H, Breier G, Knies U, Ro¨ckl W, Waltenberger J, et al. The vascular endothelial growth factor receptor Flt-1 mediates biological activities: implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 1996;271:17629–34. [31] Osaka N, Takahashi T, Murakami S, Matsuzawa A, Noguchi T, Fujiwara T, et al. ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds. J Cell Biol 2007;176:903–9. [32] Gigante B, Morlino G, Gentile MT, Persico MG, De Falco S. Plgf/ eNos/ mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia. FASEB J 2006;20:970–2. [33] Oura H, Bertoncini J, Velasco P, Brown LF, Carmeliet P, Detmar M. A critical role of placental growth factor in the induction of inflammation and edema formation. Blood 2003;101:560–7. [34] Odorisio T, Schietroma C, Zaccaria ML, Cianfarani F, Tiveron C, Tatangelo L, et al. Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability. J Cell Sci 2002;115:2559–67. [35] Adini A, Kornaga T, Firoozbakht F, Benjamin LE. Placental growth factor is a survival factor for tumor endothelial cells and macrophages. Cancer Res 2002;62:2749–52. [36] No¨r JE, Christensen J, Mooney DJ, Polverini PJ. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am J Pathol 1999;154:375–84.
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
A role of placental growth factor in hair growth Sun-Young Yoon a,b,c, Ji-Seon Yoon a,b,c, Seong Jin Jo a,b,c, Chang Yup Shin a,b,c, Jong-Yeon Shin d,e, Jong-Il Kim d,e, Ohsang Kwon a,b,c,*, Kyu Han Kim a,b,c,** a
Department of Dermatology, Seoul National University College of Medicine, Seoul, Republic of Korea Institute of Human-Environment Interface Biology, Seoul National University Medical Research Center, Seoul, Republic of Korea c Laboratory of Cutaneous Aging and Hair Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea d Genomic Medicine Institute (GMI), Medical Research Center, Seoul National University, Seoul, Republic of Korea e Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul, Republic of Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 August 2013 Received in revised form 24 January 2014 Accepted 24 January 2014
Background: The dermal papilla (DP) comprises specialized mesenchymal cells at the bottom of the hair follicle and plays a pivotal role in hair formation, anagen induction and the hair cycle. In this study, DPs were isolated from human hair follicles and serially subcultured. From each subculture at passages 1, 3, and 5 (n = 4), we compared gene expression profiles using mRNA sequencing. Among the growth factors that were down-regulated in later passages of human DP cells (hDPCs), placental growth factor (PlGF) was selected. Objective: To elucidate the effect of PlGF on hair growth. Methods: We evaluated the effect of PlGF on hDPCs and on ex vivo hair organ culture. We investigated the effect of PlGF on an in vivo model of depilation-induced hair regeneration. Results: We confirmed that the mRNA and protein expression levels of PlGF significantly decreased following subculture of the cells. It was shown that PlGF enhanced hair shaft elongation in ex vivo hair organ culture. Furthermore, PlGF significantly accelerated hair follicle growth and markedly prolonged anagen hair growth in an in vivo model of depilation-induced hair regeneration. PlGF prevented cell death by increasing the levels of phosphorylated extracellular signal-regulated kinase (ERK) and cyclin D1 and promoted survival by up-regulation of phosphorylated Akt and Bcl2, as determined by Western blotting. Conclusion: Our results suggest that PlGF plays a role in the promotion of hair growth and therefore may serve as an additional therapeutic target for the treatment of alopecia. ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Dermal papilla Gene expression profiles PlGF Hair growth
1. Introduction The hair follicle (HF) is a complicated organ, composed of multiple layers of epithelial cells and a mass of mesenchymal cells called the dermal papilla (DP) [1]. The DP is a mesenchyme-derived structure located at the bottom of the HF. The DP carries nutrients to enhance new hair growth, and contains receptors for androgens and other diverse hormones [2]. Moreover, the DP is necessary for the induction and maintenance of follicular development, mediates the hair cycle, and is thought to supply inductive signals
* Corresponding author at: Department of Dermatology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 1996; fax: +82 2 742 7344. ** Corresponding author at: Department of Dermatology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799, Republic of Korea. Tel.: +82 2 2072 2410; fax: +82 2 742 7344. E-mail addresses: [email protected] (O. Kwon), [email protected] (K.H. Kim).
required for hair outgrowth [3,4]. It is well known that abnormalities in the functions of the DP are the main causes of imbalance in follicle growth cycling and hair loss. Furthermore, the DP number dictates the size and shape of the hair, and degeneration of the DP population in mice leads to different types of hair thinning and loss [5]. However, cultured DP cells (DPCs) have been shown to exhibit significant loss of their distinct characteristics following subculture [6]. Thus, studies of specific genes related to the function of DP in the HF are necessary to further our knowledge of human hair growth. In this study, placental growth factor (PlGF) was selected as a target gene that was down-regulated in later passages of human DPCs (hDPCs). Anagen development is associated with angiogenesis, and inhibition of angiogenesis leads to suppression of anagen development [7–9]. PlGF is a member of the vascular endothelial growth factor (VEGF) subfamily and binds to VEGF receptor-1 (Flt-1), or neuropilin-1, which functions in survival, proliferation, and migration in endothelial cells, smooth muscle cells, and
0923-1811/$36.00 ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2014.01.011
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hematopoietic myeloid cells [10,11]. In adult skin, PlGF expression is up-regulated in association with both physiological and pathological neoangiogenesis, such as hair follicle cycles, wound healing, and ischemia [7,10,12–14]. In particular, PlGF is expressed in outer root sheath keratinocytes during the anagen phase of the hair follicle cycle, whereas expression of PlGF is not observed during the catagen and telogen phases [15]. While studies have demonstrated that PlGF increases cutaneous vascularization and vascular permeability, the effects of PlGF on human hair growth have not been investigated. In this study, we found that PlGF enhanced hair shaft elongation in ex vivo hair organ culture, significantly accelerated hair follicle growth, and markedly prolonged anagen hair growth in an in vivo model of depilation-induced hair regeneration. Our results demonstrated that PlGF could stimulate hair growth in both in vitro and in vivo models. 2. Materials and methods 2.1. Ethics statement This study was approved by the Institutional Review Board at the Seoul National University Hospital (approval number C-1206006-412), and all subjects provided written informed consents. All experimental procedures using human materials were conducted according to the principles described in the Declaration of Helsinki. 2.2. Isolation of human HFs and DP Skin biopsy specimens were obtained from the occipital scalp region of healthy volunteers who had not received any medication for at least 1 month. HFs were isolated under a stereo dissecting microscope, and DPs were obtained from individually isolated HFs as previously described [16,17]. HFs that were morphologically considered to be in the anagen stage were used in this study. 2.3. Cell culture The methods used for isolating and culturing hDPCs have been described previously [16]. hDPCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Welgene), 10 ng/mL of basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA), and 1 antibiotic/antimycotic solution (Gibco BRL, Gaithersburg, MD, USA), which included penicillin and streptomycin. Cells were incubated at 37 8C in a 5% CO2 incubator. 2.4. mRNA sequencing All samples were isolated from human HF tissues of different individuals. hDPs were isolated from human HFs and were serially subcultured. From each subculture at passages 1, 3, and 5 (n = 4), total RNA from hDPCs was isolated using an RNeasy Mini kit (Qiagen, GmbH, Hilden, Germany). From the mRNA-Seq sample preparation, sequencing libraries were generated according to the standard protocol of Illumina Inc. for high-throughput sequencing. We hybridized 10 pM of each library to a flow cell, with a single lane for each sample, and used an Illumina cluster station for cluster generation. Finally we generated 50-bp paired end sequences using the Illumina GAIIx sequencer (Illumina Inc., San Diego, CA, USA). We used the standard Illumina pipeline (Cassava 1.6) with default options to analyze the images and extract base calls in order to generate fastq files. The RNA sequencing reads were aligned to the NCBI human reference genome assembly (NCBI Build 36.3) using GSNAP [18] with an allowance for 5% mismatches. The expression levels for 36,742 reference sequence
(RefSeq) genes were measured by using uniquely aligned RNA sequencing reads. For each gene, the number of reads aligned to it (raw read count) was normalized by reads per kilobase per million mapped reads (RPKM) [19]. 2.5. Human HF organ culture Human scalp HFs were isolated and cultured as described previously [20,21]. Each dissected HF was cut from the bottom of the DP into a piece with a length of approximately 3.5 mm and was cultured in Williams’ E medium (Gibco BRL) supplemented with 10 ng/mL hydrocortisone, 10 mg/mL insulin, 2 mM L-glutamine, and 1 antibiotic/antimycotic solution (Gibco BRL) containing penicillin and streptomycin. Follicles were maintained at 37 8C in a 5% CO2 atmosphere. hHFs were treated with 10 or 50 ng/mL PlGF (R&D Systems, Minneapolis, MN, USA) and 1 mM minoxidil (Sigma, St Louis, MO, USA, MNX, positive control) for 8 days (n = 3). In all experiments, the tissue culture medium was changed every other day, and HF elongation was measured directly at 4 and 8 days of culture using a stereo microscope (Olympus, Tokyo, Japan). A total of 180 HFs from 3 different volunteers (60 follicles per volunteer) were analyzed in each growth condition. To evaluate the effect of anti-Flt1 neutralizing antibody, hHFs were treated with 500 or 1000 ng/mL anti-Flt1 neutralizing antibody and 1 mM MNX for 6 days (n = 4). In this experiment, the tissue culture medium was changed every third day, and HF elongation was measured directly at 3 and 6 days of culture. A total of 240 HFs from 4 different volunteers (60 follicles per volunteer) were analyzed in each growth condition. 2.6. Immunofluorescence staining Immunofluorescence staining was performed on 5-mm frozen sections of hHFs as previously described [21]. The following antibodies were used: anti-PlGF (Abcam, Cambridge, MA, USA), anti-Flt1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiKi67 (DAKO, Carpinteria, CA, USA), and anti-CD11b (BD Pharmingen, San Diego, CA, USA). Immunofluorescence images were acquired using Zeiss LSM 510 META confocal microscopy software. Confocal parameters for the pinhole, optical slide, detector gain, amplifier offset, amplifier gain, contrast, and brightness were kept constant between samples. For quantitative analyses, the number of Ki-67-positive cells was counted and normalized to the number of 40 ,6-diamidion-2-phenylindole (DAPI)-stained cells using 3dimensional software (Imaris Version 6.1, Bitplane AG, Switzerland). The number of CD11b-positive cells was counted using LAS AF Lite software (Leica Microsystems, Germany). Immunofluorescence staining for blood vessels was performed on 10-mm-thick frozen sections using anti-CD31antibody (BioLegend, San Diego, CA, USA). CD31-stained areas were measured using the Image J program (by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) as described previously [22]. To analyze apoptotic cells, we performed TUNEL labeling (In Situ Cell Death Detection Kit; Fluorescein, Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (blue fluorescence). Immunofluorescence images were obtained using the Leica Application Suite Advanced Fluorescence Software (LAS AF, Leica Microsystems, Heerbrugg, Switzerland). 2.7. Quantitative real time-polymerase chain reaction Total RNA was isolated from hDPCs using RNA iso Plus (Takara Bio Inc., Otsu, Shiga, Japan) and treated with DNase I (Roche Pharmaceuticals, Welwyn Garden City, UK) to remove genomic DNA. We used 1 mg of total RNA for the cDNA synthesis reaction,
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which was performed using a First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s instructions. To quantitatively estimate mRNA expression, polymerase chain reaction (PCR) was performed on a 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (Takara Bio Inc.) according to the manufacturer’s instructions. Primer information for human and mouse genes is given in Supplementary Tables S1 and S2. All experiments with SYBR Green were performed in triplicate and were independently repeated more than 3 times.
epidermis to the subcutaneous fat. Bulb diameter was measured in H&E-stained sections at the level of the largest diameter (‘‘Auber’s line’’) of the hair bulbs with clearly visible DP (n = 80 HFs/mouse group) [8]. For quantitative analyses, the Image J program (National Institutes of Health (NIH), Bethesda, MD, USA) was used. All animal experimental procedures were carried out at the animal facility of the Seoul National University Hospital in accordance with national and institutional guidelines.
2.8. Western blot analysis
The methods used for the anagen induction assay have been described previously [25]. The back skin of 8-week-old C57BL/6 female mice in the telogen phase was shaved with a clipper. Vehicle (phosphate-buffered saline [PBS] containing 0.1% bovine serum albumin [BSA], n = 3), PlGF (300 ng in 30 mL PBS containing 0.1% BSA, n = 4), or MNX (60 mg in 30 mL PBS containing 0.1% BSA, n = 4) were applied via intradermal injection twice every day. At 41 days, skin samples were obtained at the site of intradermal injection for histological analysis of hematoxylin and eosin (H&E)stained paraffin sections. Anagen induction scores were calculated by using assigned score values (telogen = 1, anagen I–VI = 2–7) [26–28]. The resulting values were added and divided by the number of HFs (n = 70 HFs/mouse group). In the histological analysis by H&E staining, skin thickness was measured as the distance from the epidermis to the subcutaneous fat by using the Image J program (NIH, Bethesda, MD, USA).
Total protein from hDPCs was extracted using RIPA lysis buffer (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Amersham, Buckinghamshire, UK) using a wet transfer system. The blotted membranes were incubated with the respective primary antibodies at 4 8C. The following antibodies were used: anti-total ERK, antiphosphorylated ERK, anti-total Akt, anti-phosphorylated Akt, anti-Bcl2, anti-Bax, anti-cyclin D1 (Cell Signaling Technology, Beverly, MA, USA), p53, anti-b-actin (Santa Cruz Biotechnology) and Flt1 (R&D Systems, Minneapolis, MN, USA). Membranes were probed with an anti-mouse, anti-rabbit-, or anti-goat-IgGhorseradish peroxidase conjugates (Santa Cruz Biotechnology) for 1 h at room temperature. Antibody–antigen complexes were detected using the ECL system (Amersham Pharmacia Biotech, Little Chalfont, UK). 2.9. RNA interference Knockdown of PlGF was carried out with RNA interference using small interfering RNAs (siRNAs). Control scrambled siRNA was used as a negative control siRNA (Bioneer, Daejeon, Korea). PlGF siRNA (siRNA No. 1115569) was purchased from Bioneer. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Whenever cells were transfected with PlGF siRNA, efficiency of PlGF siRNA was measured by quantitative real-time PCR. Compared with control scrambled siRNA, PlGF siRNA indicated approximately 90% efficiency. 2.10. BrdU-ELISA For the quantification of cell proliferation, a BrdU-ELISA (Roche, Mannheim, Germany) was used according to the manufacturer’s instructions. hDPCs were incubated with 10 or 50 ng/mL PlGF. After 48 or 72 h, incorporated BrdU levels were measured by ELISA and expressed as the absorbance of the treated samples relative to that of the untreated control.
2.12. Anagen hair induction test in C57BL/6 mice
2.13. Statistical analysis Statistical significance was determined using Student’s t-test. Pvalues were 2-tailed, and significance was accepted at P < 0.05. In the case of organ culture, a paired Student’s t-test was used for statistical analysis. 3. Results 3.1. mRNA and protein expression levels of PlGF significantly decreased following subculture of hDPCs DPs were isolated from human HFs and were serially subcultured. In each subculture at passages 1, 3, and 5 (n = 4), we compared the gene expression profiles of the cells using mRNA sequencing. From this gene expression analysis, we found that 232 genes were up-regulated (P < 0.01) and 2249 genes were downregulated (P < 0.01) in later passages of hDPCs. The RNA sequencing data were uploaded in EBI-SRA under accession number PRJEB5104. Among the down-regulated growth factors, PlGF was selected. We found that mRNA and protein expression levels of PlGF significantly decreased in later passages of cells (Fig. 1A and B). In addition, we also confirmed that PlGF secretion was reduced following subculture of hDPCs, as determined using enzyme-linked immunosorbent assay (ELISA; Fig. 1C).
2.11. In vivo model of depilation-induced hair regeneration 3.2. PlGF and Flt1 were expressed in hDPCs and hHFs The back skin of 8-week-old C57BL/6 female mice in the telogen phase was depilated using wax as described previously [23], resulting in synchronized induction of new anagen follicle growth. Vehicle (phosphate-buffered saline [PBS] containing 0.1% bovine serum albumin [BSA], n = 5), PlGF (300 ng in 30 mL PBS containing 0.1% BSA, n = 5), or MNX (60 mg in 30 mL PBS containing 0.1% BSA, n = 5) was applied via intradermal injection twice a day and skin samples were obtained at days 5 and 21 for histological analysis. The length of the hair follicles was measured as the distance from the bottom of the hair bulbs to the epidermis (n = 50 HFs/mouse group) [24]. Skin thickness was measured as the distance from the
We confirmed the expression of PlGF and Flt1, a PlGF receptor, by immunofluorescence staining. In hHFs, in particular, PlGF was highly expressed in the DP, and Flt1 was highly expressed in hair matrix keratinocytes (Fig. 2A and B). We also found that PlGF and Flt1 were expressed in hDPCs (Supplementary Fig. S1A and B). In addition, PlGF was expressed in human outer root sheath cells (hORSCs), which are keratinocytes of human scalp HFs (Supplementary Fig. S1C). We also observed that PlGF and Flt1 were not expressed during catagen phage of human hair follicle (Supplementary Fig. S2).
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Fig. 1. PlGF mRNA and protein levels significantly decreased in later passages of hDPCs. DPs were isolated from human HFs and subcultured (passages 1, 3, and 5). (A) Total RNA from hDPCs was isolated, and mRNA expression levels were measured by quantitative real-time PCR (q-PCR). (B) hDPCs were lysed and analyzed by Western blotting. (C) Supernatants of the different passages of hDPCs were collected, and the amount of PlGF secretion was measured by ELISA. Results are expressed as the mean standard error (SE). **P < 0.01, *P < 0.05 versus passage 1 (q-PCR and Western blot) or 3 (ELISA).
3.3. PlGF enhanced hair shaft elongation in ex vivo hair organ culture hHFs were treated with 10 or 50 ng/mL PlGF or 1 mM minoxidil (MNX), a positive control, for 8 days. We found that treatment with 50 ng/mL PlGF significantly enhanced hair shaft elongation after 8 days, as compared with that for the control group. In addition, treatment with 1 mM MNX also markedly increased hair growth after 8 days, as compared with that for the control group (Fig. 3A and B). We performed immunofluorescence staining for Ki-67, a proliferation marker, after HFs were treated with PlGF or MNX for 8 days. For quantitative analyses, the number of Ki67-positive (red fluorescence) cells was counted and normalized to the number of DAPI-stained cells (40 ,6-diamidion-2-phenylindole, blue fluorescence) in follicular matrix keratinocytes in the hair bulb. Compared with the control group, treatment with PlGF or MNX significantly increased the number of Ki-67-positive keratinocytes (Fig. 3C, P < 0.05). These results suggested that PlGF induced proliferation in hair matrix keratinocytes. In addition, treatment with an antiFlt-1 neutralizing antibody significantly reduced hair shaft elongation, as compared with that in the control group (Fig. 3D and E). These results suggested that PlGF exerted a direct effect on hair growth by binding Flt-1. 3.4. PlGF accelerated hair follicle growth and prolonged anagen hair growth We investigated whether PlGF would accelerate hair re-growth and prolong the anagen phase after depilation-induced hair regeneration. In histological analysis, measurement of hair follicle length, skin thickness, and bulb diameter is a well-known method to classify the hair cycle stages [8,24]. At day 5, the length of hair follicles was significantly increased in PlGF- and MNX-treated mice
compared with vehicle-treated mice (Fig. 4A and B, P < 0.01). The back skin of PlGF- or MNX-treated mice was thicker than that of vehicle-treated mice (Fig. 4C). These results suggested that PlGF significantly accelerated hair follicle growth and induce faster anagen entry compared with the vehicle-treated group. It is well known that catagen development induces apoptosis in hair matrix keratinocytes and leads to reduce the diameter of the hair bulb [27,29]. At day 21, hair bulbs in PlGF- and MNX-treated mice were thicker than those in vehicle-treated mice (Fig. 4D and E, P < 0.01). At day 21, to evaluate apoptotic cells, immunofluorescence staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. Compared with the control group, the PlGF- and MNX-treated groups showed a decreased number of TUNEL-positive cells (apoptosis, green fluorescence, Fig. 4F) in keratinocytes of the hair bulb. Therefore, these results suggested that PlGF markedly prolonged anagen hair growth, associated with increased thickness of hair bulbs and reduced apoptosis of hair matrix keratinocytes. To investigate whether PlGF promoted angiogenesis, immunofluorescence staining for CD31, a blood vessel marker, was performed. Compared with the vehicletreated group, the PlGF-treated group exhibited an increase in the average area of vessels (green fluorescence, Fig. 4G). PlGF has been reported to act as a strong chemoattractant for monocytes/ macrophages [30], and macrophages possess the ability to induce hair growth [31]. At day 21, to examine whether PlGF induced the recruitment of macrophages, we performed immunofluorescence staining for CD11b, a macrophage marker. Compared with the control group, the PlGF-treated group exhibited increased numbers of CD11b-positive cells (green fluorescence, Fig. 4H). Furthermore, we performed an anagen induction test, a method to evaluate the effect of PlGF on hair cycling. We could also observe skin pigmentation at the site of intradermal injection in PlGF- and
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Fig. 2. Expression of PlGF and Flt1. Immunofluorescent staining of PlGF (A, red fluorescence) and Flt1 (B, red fluorescence) was performed in hHFs. Nuclei were stained by DAPI (blue fluorescence). Original magnifications: 400, 1000.
MNX-treated group (Fig. 5A). Anagen induction score and skin thickness increased in PlGF- and MNX-treated group compared with the control group (Fig. 5B–D). These results suggested that PlGF treatment induced anagen hair cycle in telogen mouse skin. 3.5. PlGF promoted the survival of hDPCs Next, we performed Western blotting to analyze the expression of proteins related to the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) and phosphotidylinositol 3-kinase (PI3K)/Akt pathways, which play key roles in multiple cellular processes, such as apoptosis, cell proliferation, cell survival, transcription and cell migration [11]. We found that PlGF treatment markedly increased the phosphorylation of Akt and ERK and the expression of Bcl2 and cyclin D1 in a time- and dosedependent manner (Fig. 6A and B). Inhibition of Akt or ERK suppressed the mRNA expression of Flt1 and PlGF (Fig. 6C and D). These results suggested that PlGF prevented cell death by increasing the phosphorylation of ERK and the expression of cyclinD1 and promoted cell survival by up-regulating Akt phosphorylation and Bcl2 expression. Next, to elucidate the biological function of PlGF, hDPCs were transfected with PlGF siRNA for 24 or 48 h and analyzed by Western blotting. We found that suppression of PlGF decreased the phosphorylation of ERK and expression of cyclin D1. The phosphorylation of Akt was also significantly decreased (Supplementary Fig. S3A and B). In addition, suppression of PlGF dramatically increased the expression of Bax and p53, which are known to promote apoptosis (Supplementary Fig. S3A and B). Moreover, after transfection with PlGF siRNA for 48 h, we confirmed that PlGF knockdown markedly reduced the mRNA expression of cyclin D1 (Supplementary Fig. S3C). These data suggested that PlGF knockdown inhibited
proliferation through suppression of ERK and cyclin D1 and prevented survival by downregulation of Akt phosphorylation and upregulation of p53 and Bax expression. Furthermore, we also found that PlGF treatment increased the number of hDPCs and enhanced the proliferation of hDPCs, as determined by BrdU-ELISA (Supplementary Fig. S4). 3.6. PlGF enhanced the mRNA expression of some representative genes related to hair induction or proliferation of hDPCs Next, we determined the mRNA expression levels of some representative genes related to hair induction or proliferation of hDPCs. After treatment with PlGF for 30 min, mRNA expression levels of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and VEGF, which are known to stimulate hair growth, were markedly increased (P < 0.05), whereas the expression of transforming growth factor beta 1 (TGF-b1), an inhibitor of hair growth, was not changed (Fig. 6E). After treatment with PlGF for 24 h, the mRNA expression levels of versican and alkaline phosphatase (ALP), which maintain the inductive properties of the DP, significantly increased (Fig. 6F and G). In addition, mRNA expression levels of cyclin D1 and cyclin E1, markers of proliferation, increased (Fig. 6F and G). Thus, these results suggested that PlGF enhanced the expression of some representative genes related to hair induction or proliferation of hDPCs. To further support the role of PlGF in hDPCs, hDPCs were transfected with PlGF siRNA. Suppression of PlGF markedly reduced the mRNA expression levels of PlGF receptors, e.g., Flt1 and neuropilin-1, and significantly decreased the expression levels of IGF-1, HGF, fibroblast growth factor 2 (FGF-2), cyclooxygenase 2 (COX-2), and c-myc, which stimulate hair growth (Supplementary Fig. S5, P < 0.01).
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Fig. 3. PlGF enhanced hair shaft elongation in ex vivo hair organ culture. (A and B) hHFs were treated with 10 or 50 ng/mL PlGF and 1 mM minoxidil (MNX, positive control) for 8 days (n = 3). HF elongation was measured directly at 4 and 8 days of culture using a stereo microscope. (C) 5-mm frozen sections of hHFs from 3 different individuals were analyzed for proliferation (Ki67-positive, red fluorescence) in keratinocytes of the hair bulb. Nuclei were counterstained with DAPI (blue fluorescence). For quantitative analyses, the number of Ki67-positive cells was counted and normalized to the number of DAPI-stained cells. (D and E) hHFs were treated with 500 or 1000 ng/mL anti-Flt1 neutralizing antibody and 1 mM MNX for 6 days (n = 4). HF elongation was measured directly at 3 and 6 days of culture using a stereo microscope. Results are expressed as the mean SE. *P < 0.05 versus the control. Numbers next to the lines are percentage ratios compared to the control. Scale bar = 100 mm; original magnification, 200.
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Fig. 4. PlGF accelerated hair follicle growth and prolonged the anagen hair cycle. (A and D) At day 5 (A) or day 21 (D), skin samples were obtained for histological analysis of H&E-stained paraffin sections. (B and C) At day 5, the length of the hair follicles and skin thickness were measured in H&E-stained sections. (E) Bulb diameter was measured at the level of the largest diameter (‘‘Auber’s line’’) of hair bulbs. (F–H) At day 21, immunofluorescent staining for TUNEL (F, apoptosis marker, green fluorescence), CD31 (G, blood vessel marker, green fluorescence) and CD11b (H, macrophage marker, green fluorescence) was performed. Numbers on the bars are percent ratios compared to the control. Results are expressed as the mean SE. **P < 0.01 versus the control group.
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Fig. 5. PlGF induced anagen hair cycle in telogen mouse skin. (A) Hair growth was restricted to the site of intradermal injection in PlGF- and MNX-treated C57BL/6 mice. (B) Skin samples were obtained for histological analysis of H&E-stained paraffin sections. (C) Anagen induction scores were calculated and assigned score values (telogen = 1, anagen I–VI = 2–7). (D) Skin thickness was measured as the distance from the epidermis to the subcutaneous fat. Numbers on the bars are relative percentages compared to control. Results are expressed as the mean SE. **P < 0.01, *P < 0.05 versus the control group. Scale = 100 mm; Original magnification, 200.
4. Discussion In this study, PlGF was selected as one of the genes that were down-regulated in later passages of hDPCs. Hair matrix keratinocytes are known to be important for hair growth, and the DP regulates the activity of matrix keratinocytes [6]. We confirmed that PlGF and Flt1, a PlGF receptor, were expressed in hDPCs and HFs. Furthermore, we found that PlGF increased the proliferation of human hair matrix keratinocytes, as determined by immunofluorescence staining for Ki-67, a proliferation marker. Interestingly, in this study, we found that PlGF enhanced hair shaft elongation in ex vivo hair organ culture, whereas hair growth was inhibited by blockade of Flt-1, a PlGF receptor. These results suggested the possibility that PlGF, a paracrine factor secreted from DPCs, acted on neighboring follicular matrix keratinocytes and had a direct stimulatory effect on hair growth by binding Flt-1. It has been reported that PlGF is expressed in ORS during the angiogenic phase of the hair follicle cycle [7,15] and we also observed that PlGF was expressed in hORSCs, which are keratinocytes of human scalp hair follicle. To investigate a paracrine effect of PlGF a little more deeply, further studies need to be undertaken to explore the effect of PlGF on hORSCs, which are keratinocytes of hHFs. Some reports have indicated that PlGF-deficient mice or PlGFtransgenic mice was associated with vascularization and vessel permeability but did not show a certain noticeable changes in hair phenotypes [12,32,33]. Otherwise, in our study, in an in vivo model of depilation-induced hair regeneration, PlGF significantly accelerated hair follicle growth, and markedly prolonged anagen hair growth. Furthermore, through an anagen hair induction test in C57BL/6, we could confirm that PlGF treatment induced anagen hair growth in telogen mouse skin due to the promoting effect on the hair cycling. Overexpression of PlGF strongly increases vascularization and enhances vessel permeability [33,34]. Additionally, perifollicular angiogenesis has been shown to be correlated with up-regulation of VEGF expression in follicular keratinocytes of the outer root sheath during the anagen phase [8]. Transgenic overexpression of VEGF improves perifollicular vascularization and accelerates murine hair regrowth after depilation. In the current study, we
showed that PlGF could increase the average area of vessels. Thus, this increase in angiogenesis by PlGF may play a role in improving hair growth. In this study, we also found that PlGF induced the recruitment of macrophages. PlGF is a strong chemoattractant for monocytes/ macrophages [30], and the increased expression of PlGF in K14PlGF diabetic wounds correlates with enhanced monocyte/ macrophage recruitment at the wound site [12]. According to Osaka et al., macrophages possess the ability to induce hair growth [31]. Therefore, their recruitment by PlGF is suggestive of its role as a chemotactic factor and it may have an indirect stimulatory effect on hair growth via macrophage. In human keratinocytes, mRNA and protein expression of PlGF were increased by epidermal growth factor (EGF), TGFa, and TGFb, key cytokines in wound healing, as well as interleukin 6 (IL-6), an inflammatory cytokine [13]. mRNA levels of PDGF, FGF-2 and VEGF, which play roles in wound healing, have been reported to show a significant increase in wounds of diabetic mice, following transfection with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) [12]. Our study also demonstrated that in hDPCs, PlGF treatment significantly increased mRNA expression of IGF-1, HGF, and VEGF, which are thought to stimulate hair growth, whereas the mRNA level of TGF-b1, an inhibitor of hair growth, was not changed. Some reports have indicated that PlGF promoted the survival function of endothelial cells via induction of the expression of the anti-apoptotic gene, survivin [35]. Moreover, in human dermal microvascular endothelial cells, survival was enhanced by induction of Bcl-2 expression [36]. In Western blot analysis, we observed that in hDPCs, PlGF increased the expression of Bcl-2, which functions in survival. Furthermore, we also found that PlGF clearly increased the protein expression of cyclin D1 and the phosphorylation of ERK and Akt, which are known to promote survival and prevent cell death. In addition, PlGF treatment increased the number of hDPCs and enhanced the proliferation of DPCs, as determined by Brdu-ELISA. Taken together, these results showed that PlGF secreted from hDPCs may exhibit an autocrine function on hDPCs. In summary, our results demonstrated that PlGF markedly stimulated hair growth in both in vitro and in vivo models. We
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Fig. 6. PlGF promoted the survival of hDPCs. (A and B) hDPCs were incubated with the indicated concentrations of PlGF. After 24 (A) or 72 h (B), cells were lysed and analyzed by Western blotting. (C and D) hDPCs were treated with 1 mM LY294002 (an inhibitor of Akt, C) or PD98059 (an inhibitor of ERK, D) for 48 (C) or 24 h (D). Total RNA was isolated and mRNA expression was analyzed by quantitative real-time PCR. (E and F) Cells were treated with 50 ng/mL PlGF for 30 min (E) or 24 h (F), total RNA was isolated, and mRNA expression was analyzed by quantitative real-time PCR. (G) hDPCs were treated with the indicated concentrations of PlGF for 24 h, total RNA was isolated, and mRNA expression was analyzed by RT-PCR. Results are expressed as the mean SE. *P < 0.05 versus the control. ALP, alkaline phosphatase.
propose that PlGF plays an important role as an inducer of hair follicle growth and, therefore, may serve as an additional therapeutic target for the treatment of alopecia. Funding sources This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (Grant No.: A103017); by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (No.: 2012R1A1A2005302); partially by a research agreement with AmorePacific Corporation, Republic of Korea.
Acknowledgements We thank Elizabeth Park, Boston University School of Medicine for her assistance during the manuscript preparation. This study was presented at the 7th World Congress for Hair Research in Edinburgh, Scotland, UK, in May 2013. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.01.011.
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