Biomolecules & Therapeutics 2019; 27(4): 404-413  https://doi.org/10.4062/biomolther.2018.195
Udenafil Induces the Hair Growth Effect of Adipose-Derived Stem Cells
Nahyun Choi1,2,*, and Jong-Hyuk Sung1,2,*
1College of Pharmacy, Yonsei University, Incheon 21983, Republic of Korea, 2STEMORE Co. Ltd., Incheon 21984, Republic of Korea
*E-mail: brian99@empal.com (Sung JH), nh147837@gmail.com (Choi N), Tel: +82-32-749-4506 (Sung JH), +82-32-749-4165 (Choi N)
Received: October 8, 2018; Revised: January 1, 2019; Accepted: February 25, 2019; Published online: April 10, 2019.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Udenafil, which is a PDE5 inhibitor, is used to treat erectile dysfunction. However, it is unclear whether udenafil induces hair growth via the stimulation of adipose-derived stem cells (ASCs). In this study, we investigated whether udenafil stimulates ASCs and whether increased growth factor secretion from ASCs to facilitate hair growth. We found that subcutaneous injection of udenafil-treated ASCs accelerated telogen-to-anagen transition in vivo. We also observed that udenafil induced proliferation, migration and tube formation of ASCs. It also increased the secretion of growth factors from ASCs, such as interleukin-4 (IL-4) and IL12B, and the phosphorylation of ERK1/2 and NFκB. Furthermore, concomitant upregulation of IL-4 and IL12B mRNA levels was attenuated by ERK inhibitor or NFκB knockdown. Application of IL-4 or IL12B enhanced anagen induction in mice and increased hair follicle length in organ culture. The results indicated that udenafil stimulates ASC motility and increases paracrine growth factor, including cytokine signaling. Udenafil-stimulated secretion of cytokine from ASCs may promote hair growth via the ERK and NFκB pathways. Therefore, udenafil can be used as an ASC-preconditioning agent for hair growth.

Keywords: Udenafil, Adipose-derived stem cells, IL-4, IL12B, Hair growth
INTRODUCTION

Adipose-derived stem cells (ASCs) are multipotent cells that not only differentiate into mesenchymal lineage cells but also secrete various growth factors thereby promoting hair growth (Bunnell et al., 2008; Won et al., 2010; Festa et al., 2011; Jeong et al., 2013b; Kim et al., 2014a, 2014b, 2015; Jin and Sung, 2016). ASCs release multiple growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factors (b-FGF) which induce the proliferation of dermal papilla cells (DP cells), thereby promoting hair growth (Won et al., 2010). The lineage experiment revealed that premature adipocyte drives hair cycling as skin stem cell niche releasing platelet-derived growth factor-A (PDGF-A) to regulate follicular stem cell activity (Festa et al., 2011). ASC transplantation promotes hair growth in vivo, and a conditioned medium of ASCs increased the proliferation of hair-compositing cells in vitro (Jin and Sung, 2016). Therefore, transplantation of ASC-enriched adipose tissue has shown promise as an alternative approach to treating hair loss in men and women (Perez-Meza et al., 2017). However, our many trials have found that only ASC or conditioned medium of ASCs is not enough to promote hair growth significantly and effectively. Therefore, ASC preconditioning with identified stimulators is one of the best strategy that can be used to enhance the efficacy of ASCs.

We have identified stimulators that enhance the hair growth potential of ASCs in vivo. For instance, vitamin C and low-dose UVB increased secretion of hair growth-promoting factors from ASCs and induced the rate of anagen to telogen in animal models (Jeong et al., 2013b; Kim et al., 2014a). Platelet-derived growth factor-D (PDGF-D) revealed strong effects on ASCs and increased secretion of growth factors via mitogen-activated protein kinase (MAPK) pathways and ROS generation in mitochondria (Kim et al., 2015). LL-37 increased the secretion of growth factors and the hair growth efficacy of ASCs via early growth response 1 (EGR1) protein and the MAPK pathway (Yang et al., 2016). The vasodilator, such as minoxidil, are known to stimulate hair growth (Buhl et al., 1989; Headington, 1987; Michelet et al., 1997; Li et al., 2001; Kwack et al., 2011; Otomo, 2002; Han et al., 2004). However, the exact mechanism for hair growth of vasodilators has not yet to be understood. The general hypothesis is that the increase in blood flow causes dormant follicles to reconnect with the dermal papillae, and this structures located under the follicle provide a blood supply to the hair bulb. There is strong evidence that minoxidil directly promotes hair growth via stimulation of DP cells and epithelial cells (Han et al., 2004). In addition, we found that preconditioned ASCs with minoxidil promoted hair growth releasing growth factors including PDGF-C, PD-ECGF and CXC1 via ERK pathway (Choi et al., 2018). Therefore, we next wanted to examine hair promoting effect of other vasodilators such udenafil.

Udenafil (Dong-A, Seoul, Korea), a newly introduced drug in urology, is used to treat erectile dysfunction. It belongs to a class of drugs called phosphodiesterase type 5 (PDE5) inhibitors, which also include avanafil, sildenafil, tadalafil, and vardenafil. Udenafil acts by inhibiting the degradation of cyclic guanosine monophosphate (cGMP) to guanosine monophosphate (GMP), which relaxes the smooth muscle and allows increased blood flow for penile tumescence (Gopal et al., 2001). Previous studies have proven that increased cGMP levels due to an active PDE5 inhibitor induces angiogenesis by forming capillary-like tubes in the corpus cavernosum and increasing VEGF synthesis (Pyriochou et al., 2007; Sahara et al., 2010). Preconditioning of PDE5 inhibitors with ASCs improves the therapeutic efficacy of ASCs in myocardial infarction in vivo, releasing growth factors, including VEGF, b-FGF, and insulin-like growth factor (IFG) (Hoke et al., 2012). In a previous study, chronic administration of udenafil promoted erectile function in a BCNI rat model and suppressed ameliorating fibrosis and apoptosis induced by cavernous nerve crush injury (Lee et al., 2011). Udenafil improves penile reaction in diabetic animals, animals with spinal cord injury and hypertensive patients without increasing the frequency or severity of adverse events (Kang et al., 2004; Paick et al., 2009). In addition, in combination with covering ASCs, udenafil can improve erec-tile dysfunction (Jeong et al., 2013a).

However, there is little evidence that preconditioning of udenafil on ASCs can affect hair growth. Therefore, in this study, we examined the preconditioning effect of udenafil in ASCs and growth factor secretion enhancing hair growth.

MATERIALS AND METHODS

Cell culture

Human ASCs were isolated via liposuction of subcutaneous fat, as described in a previous study (Kim et al., 2007; Yi et al., 2014): Briefly, the fat was washed with phosphate-buffered saline (PBS), 0.075% collagenase was added and the mixture was incubated for 45 min. at 37°C with gentle shaking. After centrifugation, the pellet was filtered through a 100 μm nylon mesh. Then, the cells were cultured with an essential medium including alpha Minimum Essential Medium (α-MEM) (Hyclone, Logan, UT, USA), 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% antibiotics (Gibco) for three passages. Then, the medium was changed to α-MEM, 10% FBS and 1% penicillin/streptomycin (Gibco) for four passages. ASCs were used at passages 5-7 for all experiments. Characterization of ASCs was performed using flow cytometry. ASCs were positive for CD44, CD73, CD90, CD105, human leukocyte antigen (HLA)-I, and podocalyxin-like protein (PODXL) but were negative for hematopoietic markers such as CD34 and CD45 (Kim et al., 2007). The multipotent differentiation potential was examined, as described in a previous study (Yi et al., 2014), and ASCs could be differentiated into adipocytes, osteocytes, and chondrocytes. ASCs were maintained at 37°C in a humidified 5% CO2 incubator.

Cell growth assay

For cell growth assay, ASCs were seeded in 12-well plates, with 5×103 cells/well, treated with udenafil (0.1, 0.5 or 1 μM) and incubated for 7 days. ASCs were also seeded in 12-well plates, with 1×104 cells/well, treated with synthetic peptides, IL-4 or IL12B (100 ng/mL) and incubated for 7 days. The cells were then trypsinized, stained with trypan blue (Sigma-Aldrich, MO, USA) and counted each day using a hemocytometer.

Scratch wound-healing assay

ASCs were seeded into 6-well plates and cultured to confluence. A sterile 1 mL pipette tip was used to scratch the cell monolayer. The cultures were then washed with PBS to remove de-plated cells, and the cells were again cultured with udenafil, IL-4, or IL12B in a serum-free medium for 3 days. Cell migration into the scratched area (wound closure) was visualized using a ZEISS Observer.D1 microscope. Multiple images were acquired per well, and the average cell number within the wound were monitored over 3 days.

Transwell migration assay

ACSs were seeded into 60 mm plates and treated with udenafil, IL-4, or IL12B for 3 days. ASCs starved for 1 day (1.5×104 cells/well) were suspended in a serum-free medium and seeded on the upper side of transwell membrane insert (BD Falcon, CA, USA), which was pre-coated with matrigel (1/60 dilution, BD Matrigel matrix, CA, USA). The normal serum with FBS was added in lower plate as chemoattractant. The cultures were incubated for 1 day to allow transwell migration. The inserts were then removed, and their upper surface was cleaned using cotton swabs and washed with PBS to remove non-migrating cells. The inserts were stained with 0.1% formalin/10% crystal-violet solution (Sigma-Aldrich) for 20 min, and cell number was analyzed under a ZEISS Observer.D1 microscope. Multiple images (15–20) were acquired per insert, and the average cell counts were calculated.

Tube formation assay using matrigel

For this experiment, 12-well plates were coated with matrigel (1/2 dilution, BD Matrigel matrix) and dried for 2 h at 37°C. ASCs treated with endothelial cell basal medium-2 (EBM-2, LONZA, MD, USA) and udenafil were plated in matrigel-coated wells and incubated for 16 h at 37°C. The number of tubes was analyzed under a ZEISS Observer.D1 microscope.

RNA extraction, cDNA synthesis, QPCR and QPCR array

Total RNA was extracted from ASCs using Trizol reagent (Invitrogen, NY, USA) and was subjected to complementary DNA (cDNA) synthesis using oligodT and the HelixCriptTM Thermo Reverse Transcription System (NANOHELIX, WI, USA) according to the manufacturer’s instructions. Bright-Green QPCR master mix-ROX (abm, NY, USA) was used for QPCR reactions. For the QPCR array, total RNA was extracted from ASCs or 1 μM udenafil-treated ASCs and subjected to cDNA synthesis, as described above. QPCR array for growth factors was conducted using an RT2 First Strand cDNA Synthesis Kit (QIAGEN, MD, USA).

Western blot

For western blotting of phospho-ERK, ASCs were treated with udenafil for 15 or 30 min. Next, the cells were treated with U0126 (10 μM, CALBIOCHEM, CA, USA) for 1 h and lysed with protein extraction solution (PRO-PREPTM; iNtRON, Seoul, Korea) containing a phosphatase inhibitor (Na3VO4; Roche, CA, USA). Western blot analysis was performed as follows. Briefly, protein extracts were loaded on the acrylamide gel, blotted on the NC membrane, incubated with the following primary antibodies: mouse anti-phospho-p42/44 (1:1500; Cell Signaling Technology, MA, USA), and mouse anti-α-tubulin (1:2000; Santa Cruz Biotechnology, TX, USA) overnight at 4°C. Next, the membrane was incubated with HRP-tagged secondary antibodies (Jackson ImmunoResearch, PA, USA) for 1 h and blot images were obtained using ImageQuant LAS 4000 (GE Healthcare Life Science, PA, USA). For western blot of phospho-NFκB, siRNA for negative control or NFκB were treated for 2–3 days, then udenafil was treated for 15 or 30 min treated and were lysed with protein extraction solution. Western blot analysis was performed using the following primary antibodies, rabbit anti-NFκB (1:1000; Santa Cruz Biotechnology) and blot images were obtained using ImageQuant LAS 4000.

Animal experiment

Mice were maintained and anesthetized according to a protocol approved by the US Pharmacopoeia and the Institutional Animal Care and Use Committee of Yonsei University (IACUCA-201802-183-01). The dorsal area of 6.5-week-old C3H/HeN mice in the telogen stage of the hair cycle was shaved with an electric shaver. Naïve ASCs or 1 μM udenafil-treated ASCs (3×104 cells/mice) were injected once into the dorsal skin of the shaved mice for 14 days. IL-4 or IL12B protein (1 μg/mL/day; PeproTech, NJ, USA) was applied on the dorsal skin of the shaved mice at 6- week-old every day for 16–17 days. The skin darkening (indicative of anagen cycle induction) was carefully monitored by photography. After that, the dorsal hair was shaved and weighted in order to estimate the growth rate (Buhl et al., 1989)

Vibrissae follicle organ culture

For organ culture of vibrissae hair follicles, we cut the vibrissae hair follicles from 4 weeks c57bl/6 mice, washed them with PBS and cultured them in a defined medium (williams E medium supplemented with 2 mM L-glutamine, 10 μg/mL insulin, 10 ng/mL hydrocortisone, 100 U/mL penicillin and 100 μg/mL streptomycin, without a serum) including IL-4 or IL12B (5 and 20 ng/mL) for 3 days at 37°C in a humidified 5% CO2 incubator. Images were obtained using Nikon SMZ800N microscope (Nikon, Tokyo, Japan) at 0 and 3 days, and analyzed using Adobe Photpshop CS6 extended program (Adobe, CA, USA).

HE and immunofluorescence staining

HE staining, paraffin sections were de-waxed using xylene for 30 min, hydrated in 100%, 90%, 80% and 70% ethyl alcohol (EtOH), dipped into Mayer’s hematoxylin (Sigma-Aldrich) for 8 min, and then rinsed in water for 10 min. The slides were again dipped into eosin Y (Sigma-Aldrich) for 80 s, dehydrated with 70%, 80%, 90% and 100% EtOH, washed with fresh xylene for 30 min and dried and mounted with a mounting medium. Immunofluorescence staining was performed using standard protocols: Briefly, paraffin sections were de-waxed using xylene for 30 min. and hydrated in 100%, 90%, 80%, and 70% EtOH, and antigen retrieval was performed by boiling the sections in an antigen retrieval buffer using a microwave (Dako, CA, USA) for 2 min 20 sec. The sections were then treated with rabbit Ki67 antibody (1:300) (Abcam, Cambridge, UK) overnight at 4°C and then incubated with secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Immunofluorescence staining was performed and images obtained using a ZEISS LSM700 confocal microscope (Ca Zeiss, Oberkochen, Germany).

Statistical analysis

All experiments were performed more than three times using independent cultures. Data were presented as mean ± standard error of the mean. The means were compared by Student’s t-test. For all statistical tests, a 0.05 level of confidence was accepted as being statistically significant.

RESULTS

Udenafil-pretreated ASCs induce cell proliferation and migration

Although ASCs secrete various growth factors that promotes hair growth (Won et al., 2010; Festa et al., 2011; Jeong et al., 2013b; Kim et al., 2014a, 2015; Jin and Sung, 2016), it is still unmet that promotion of hair growth using preconditioning of ASCs with trichogen. First, we investigated the effect of udenafil on growth and migration of human ASCs. We counted of the live cell number for 7 days after udenafil treatment and found that udenafil increases ASC growth in a time- and dose-dependent manner (Fig. 1A). To investigate the effect of udenafil on ASC migration, we conducted transwell migration assay using insert and scratch wound-healing assay. The results showed that udenafil increases ASC migration into both assays in a dose-dependent manner (Fig. 1B–1D). Initially, it was believed that application of preconditioned udenafil in ASCs stimulates vasodilatation and increases blood flow by inhibiting the cGMP degradation (Gopal et al., 2001). Therefore, we examined whether udenafil affects blood vessel formation in ASCs. The result showed that udenafil increases the number of nascent tubes after 16 h (Fig. 1E), indicating an increase in the ASC tube-forming activity. Collectively, these results suggested that udenafil may promote hair growth by enhancing ASC proliferation and migration. Considering the previous reports that sildenafil enhances melanoma growth by promoting MAPK signaling (Dhayade et al., 2016), we examined the effect of shildenafil which is one of the other PDE5 inhibitors on ASCs growth and migration. The various concentration of shildenafil couldn’t effect on ASCs growth and migration (Supplementary Fig. 1), suggesting that shildenafil might not be stimulator of hair growth due to no effect on ASCs growth and migration.

Udenafil-pretreated ASCs promote hair growth in vivo

When we investigated the hair growth effect of udenafilpretreated ASCs in vivo, we found that subcutaneous injection of untreated ASCs (ASCCtrl) slightly increased the rate of hair growth in mice, while udenafil-pretreated ASCs (ASCudenafil) induce marked the rate of hair growth (Fig. 2A). To examine the effect of udenafil-pretreated ASCs on hair follicles, we performed hematoxylin and eosin (HE) staining and immunofluorescence staining for Ki67, a proliferating cortex cell marker. The skin section of ASCudenafil-treated mice showed a higher number of mature hair follicles compared to vehicle- or ASCCtrl-treated mice (Fig. 2B). In addition, most hair follicles of ASCudenafil-treated mice showed cortex region with Ki67+ cells compared to vehicle- or ASCCtrl-treated mice (Fig. 2C). These results indicated that most hair follicle cycling of ASCudenafil-treated mice is in anagen phage compared to vehicle- or ASCCtrl-treated mice. All results suggested that udenafil-preconditioned ASCs can promote telogen-to-anagen induction, thereby promoting hair growth.

IL-4 and IL12B induce hair growth

It has been reported previously that growth factors secreted by ASCs, such as VEGF, b-FGF, PDGF-A, PDGF-D chemokine (C-X-C motif) ligand 1 (CXCL1), PD-ECGF, and PDGF-C, regulate hair follicular stem cell activity and induce the anagen phase of the hair cycle in vivo (Won et al., 2010; Festa et al., 2011; Kim et al., 2015; Choi et al., 2018). In addition, preconditioning of PDE5 inhibitors with ASCs improves the efficacy of ASCs in myocardial infarction, releasing VEGF, b-FGF, and IFG (Hoke et al., 2012). We hypothesized that udenafil may indirectly promote hair growth by enhancing growth factor release from ASCs. Therefore, we compared the expression pattern of cell growth-related genes between untreated and udenafil-treated ASCs by qPCR array (Fig. 3A). After QPCR array analysis, we found that the expression of many growth factor genes were upregulated in udenafil treated ASCs compared to control (Supplementary Fig. 2). Among top-notch genes, we selected 6 genes and confirmed their expression by QPCR. We finely selected interleukin 4 (IL-4) and subunit beta of interleukin (IL12B) as potential target genes of udenafil because their consistent upregulation was confirmed by QPCR (Fig. 3B). To investigate whether IL-4 and IL12B can induce the telogen-to-anagen phase of the hair cycle faster than the control in vivo, we applied human IL-4 or IL12B protein on the back skin of shaved mice. We observed that the application of both IL-4 and IL12B proteins significantly induced the telogen-to-anagen phase of the hair cycle and increased the number of mature hair follicles and Ki67+ hair follicles (Fig. 3C), suggesting that udenafil may induce anagen by triggering IL-4 and IL12B release from ASCs. Moreover, treatment with IL-4 and IL12B also increased the length of isolated mouse vibrissal hair follicles in organ culture (Fig. 3D). These results strongly suggested that udenafil promotes hair growth through the release of cytokines such as IL-4 and IL12B from ASCs.

IL12B induce ASC proliferation and migration, but IL-4 induces migration

To examine whether IL-4 and IL12B affect ASC proliferation, we followed the live cell number for 7 days after IL-4 or IL12B treatment. We found that IL12B, not IL-4, increased ASC proliferation in a time- and dose-dependent manner (Fig. 4A). Furthermore, to explore whether IL-4 and IL12B affect ASC migration, we conducted transwell migration and scratch wound-healing assays. Both assays showed that IL-4 and IL12B increased ASC migration dose-dependently (Fig. 4B–4E). Collectively, these results suggested that IL-4 and IL12B may promote hair growth by enhancing ASC migration and partial proliferation. To clarify how increased IL-4 and IL12B in udenafil-treated ASCs is functioned in hair-compositing cells such as dermal papilla cells (DP cells), we examined the effect of IL-4 and IL12B on DP cells. Indeed, the treatment of each IL-4 or IL12B protein increased growth of DP cells (Supplementary Fig. 3). This result suggested that released IL-4 or IL12B from ASCs increased growth of DP cells thereby stimulating hair growth.

Udenafil regulates IL4 and IL12B expression in ASCs via the ERK pathway

Previous studies have reported that the MAPK pathway regulates growth factor expression in ASCs, including VEGF-A and FGF2 (Kim et al., 2014a, 2014b). Therefore, in this study, we examined whether udenafil regulates IL-4 or IL12B expression via the MAPK pathway. We found that udenafil upregulated ERK phosphorylation time-dependently, a response suppressed by U0126, a specific MEK inhibitor (Fig. 5A). U0126 suppressed udenafil-increased proliferation and migration of ASCs (Fig. 5B, 5C) and reversed udenafil-induced IL-4 and IL12B upregulation (Fig. 5D). These results suggested that udenafil upregulates IL-4 and IL12B expression in ASCs via the MAPK pathway.

Udenafil regulates IL4, not IL12B, expression in ASCs via the NF-κB pathway

Previous studies have reported that nuclear factor kappa B (NFκB) inhibition suppresses both IL-4 and IFN-dependent increase in polymeric immunoglobulin receptor (pIgR) expression in human intestinal epithelial cells (HT29), suggesting immunity regulation (Ackermann and Denning, 2004). Therefore, in this study, we examined whether udenafil regulates the IL-4 or IL12B expression via the NFκB pathway. We found that udenafil upregulated NFκB phosphorylation at15 min post-treatment, and this response was suppressed by NFκB knockdown, which was confirmed by messenger RNA (mRNA) and protein down-regulation (Fig. 6A, 6B). Furthermore, NFκB knockdown recovered udenafil-increased proliferation and migration in ASCs (Fig. 6C, 6D). Finally, NFκB knockdown also reversed udenafil-induced upregulation of IL-4 but not IL12B (Fig. 6E). These results suggested that udenafil upregulates IL-4, not IL12B, expression in ASCs via the NFκB pathway.

DISCUSSION

Udenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5, is used to treat erectile dysfunction. However, it was not understood the link between udenafil and hair promoting effects through ASC stimulation. Therefore, in this study, we investigated whether udenafil stimulates ASCs and enhances hair growth through growth factor release. First, we demonstrated that subcutaneous injection of udenafil-preconditioned ASCs accelerates telogen-to-anagen transition in vivo, increasing the number of proliferating hair follicles as well as hair weight. Second, udenafil increases ASC proliferation, migration, tube formation and secretion of cytokines such as IL-4 and IL12B via ERK or NFκB. Third, application of these cytokines enhances anagen induction in mice and also increases the growth of isolated mouse vibrissal hair follicles in organ culture.

IL-4 is a cytokine that induces activated B-cell and T-cell proliferation and naïve helper T-cell differentiation, indicating it is a key regulator in humoral and adaptive immunity (Bao and Reinhardt, 2015). Although the role of IL-4 in hair growth or alopecia areata (AA) has not been reported yet, there is evidence of the relationship between AA and cytokines (Hoffmann, 1999; Gregoriou et al., 2010). AA is a highly prevalent, chronic, and relapsing autoimmune disease that targets hair follicles. IL18 is a significant pro-inflammatory cytokine that is present in high levels in AA patients (Celik and Ates, 2018). Recent data have suggested the possible role of IL17 in AA pathogenesis (Ramot et al., 2018). In addition, IL1 is a potent inducer of hair loss and a significant inhibitor of human hair growth in vitro (Hoffmann and Happle, 1995). Also, Groves et al. (1994) have shown that transgenic mice over-expressing IL1a in the epidermis have patchy hair loss resembling AA. IL-4-mediated apoptosis may participate in catagen formation regulation in hair follicles, acting selectively on cultured keratinocyte and being independent of bcl-2 and bax expression (Mandt et al., 2002). There is no evidence of the relationship of another cytokine, IL12B, which acts on T- and natural killer (NK) cells, with hair growth. Studies have only reported that cytokines such as IL12B, IL10, IL36RN, IL6, IL2, IL23, IL2RA, and IL4R are related to autoimmune diseases (Redler et al., 2012).

Although ASC transplantation promotes hair growth in vivo experimentally and ASC-CM induces the proliferation of hair-compositing cells in vitro, ASCs and ASC-CM have shown limited effectiveness on hair growth. In addition, our many animal experiments revealed that only ASC or ASC-CM injection is not enough to promote hair growth significantly. ASC preconditioning is one strategy that can be used to enhance ASC efficacy. Our group has already shown preconditioning methods of enhancing hair growth using ASC stimulators, such as vitamin C, PDGF, hypoxia, UVB, and minoxidil (Kim et al., 2011; Jeong et al., 2013b; Kim et al., 2014a, 2015). Especially, preconditioned ASCs with minoxidil, which is widely used for AA treatment, induce hair follicle cycling and hair growth-releasing growth factors, including PDGF-C (Choi et al., 2018). In addition to hair loss treatment, minoxidil is also used as a vasodilator. Studies have reported that minoxidil induces the tube-forming activity of ASCs and expression of angiogenic-related genes (Choi et al., 2018). Udenafil is also used as a vasodilator and also increases the tube-forming activity, proliferation and migration of ASCs (Fig. 2A) suggesting that udenafil may be used as a stimulator of ASCs to enhance hair growth. Although the role of other PDE5 inhibitors, such as avanafil, sildenafil, tadalafil, and vardenafil, in hair growth has not been investigated, it would be meaningful to investigate this function of theirs. Indeed, the use of a PDF5 inhibitor such as sildenafil in preconditioned ASCs improved myocardial infarction by releasing growth factors (VEGF, b-FGF, and IGF) (Hoke et al., 2012). However, as we examined the effect of shildenafil on ASCs growth and migration, various concentration of shildenafil couldn’t effect on ASCs growth and migration (Supplementary Fig. 1). Although preconditioned ASCs with sildenafil can release some growth factors, it didn’t stimulate ASCs. This means that shildenafil might not be stimulator of hair growth using ASCs.

We also investigated the increased cellular phenotype and upregulation of IL4 and IL12B by udenafil via the MEK-ERK and NFκB pathways. However, udenafil acts by inhibiting the degradation of cGMP to guanosine monophosphate (GMP), which relaxes the smooth muscle and allows increased blood flow for penile tumescence (Gopal et al., 2001). Smooth muscle relaxation is, in part, mediated via protein kinase G (PKG) activation, subsequent potassium channel opening and reduction in intracellular calcium levels. Although we did not investigate the relationship between the cGMP-PKG pathway and hair growth in this study, it will be done in another project.

In summary, subcutaneous injection of udenafil-preconditioned ASCs accelerates telogen-to-anagen transition in mice, and direct udenafil treatment increases tube formation, proliferation, migration, and cytokine secretion by ASCs. The most strongly upregulated cytokines, IL-4 and IL12B, individually enhance anagen induction in mice and also increase the growth of isolated mouse vibrissal hair follicles in organ culture. In addition, upregulation of IL-4 by udenafil are mediated via both ERK and NFκB pathways, and upregulation of IL12B is mediated by ERK pathway. Therefore, udenafil can be used as a novel ASC-preconditioning agent for hair regeneration (Fig. 7).

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare and have not received any payment for the preparation of this manuscript.

ACKNOWLEDGMENTS

This study was supported by a grant from the National Research Foundation (NRF-2016R1D1A1B03932050). NC was also supported by the National Research Foundation (NRF-2017R1A6A3A11035599) funded by the Korean government.

SUPPLEMENTARY
Figures
Fig. 1. Udenafil promotes ASC proliferation and migration. (A) Cell growth was measured after udenafil treatment in ASCs for 7 days. (B–D) Cell migration was measured after udenafil treatment using transwell migration assay (B) and scratch wound-healing assay (C, D). (E) Tube formation assay was carried out to show the enhanced tube formation by udenafil-treated ASCs. *p<0.05; **p<0.01; ***p<0.001. Three independent experiments were conducted per all data point. All error bars indicate SEM.
Fig. 2. Udenafil-pretreated ASCs promote hair growth in vivo. Udenafil-treated ASCs or untreated ASCs were injected into the dorsal skin of shaved mice. Photographs were taken and the hair weight measured (A) 14 days later. (B) A skin section was analyzed by HE staining, and the number of mature hair follicle was measured. (C) A hair follicle with Ki67+ cells in cortex region is shown by immunostaining. Asterisks indicate hair follicles with Ki67+ cortex cells. *p<0.05; **p<0.01; ***p<0.001. N=6 or 7 mice per group. All error bars indicate standard error of the mean (SEM).
Fig. 3. Udenafil upregulates the expression of hair growth-promoting factors IL-4 and IL12B in ASCs. (A) Upregulation of many growth factors in udenafil-treated ASCs compared to untreated ASCs was analyzed by QPCR array for growth factors. Pink straight lines indicate ± 2 fold change, and arrows indicate the two genes, IL-4 and IL12B, analyzed in (B). (B) Confirmation of six upregulated genes, IL12B, FGF6, BMP10, IL-4, BMP7 and EREG, between untreated and udenafil-treated ASCs using QPCR. (C) Human IL-4 and IL12B proteins each facilitate hair growth and increase the number of mature proliferating hair follicle after application on the dorsal skin of shaved mice. N=7-8 mice per group. (D) IL-4 and IL12B proteins each facilitate mouse vibrissal hair follicle growth. N=10 samples per treatment group. *p<0.05; **p<0.01; ***p<0.001. All error bars indicate SEM.
Fig. 4. IL12B induce ASC proliferation and migration, but IL-4 induces migration (A) Cell growth was measured after treatment by IL-4 or IL12B in ASCs for 7 days. Three independent experiments were conducted. (B-E) IL-4 or IL12B enhances ASC migration, as evidenced by both (B, C) transwell migration and (D, E) scratch wound-healing assays. *p<0.05; **p<0.01; ***p<0.001. Three independent experiments were conducted per data point. All error bars indicate SEM.
Fig. 5. Udenafil upregulates IL-4 and IL12B expression through the ERK pathway in ASCs. (A) The MEK inhibitor U0126 reversed udenafil-induced ERK phosphorylation. (B, C) U0126 suppressed udenafil-increased growth and migration of ASCs. (D) U0126 also suppressed udenafil-induced upregulation of IL-4 and IL12B expression in ASCs. Three independent experiments were carried out per every experiment. **p<0.01; ***p<0.001. All error bars indicate SEM.
Fig. 6. Udenafil upregulates IL-4, not IL12B, expression in ASCs via the NFκB pathway. (A) NFκB knockdown reversed udenafil-induced NFκB phosphorylation. (B) NFκB down-regulation was confirmed by QPCR. (C, D) NFκB knockdown suppressed udenafil-increased growth and migration of ASCs. (E) NFκB knockdown also suppressed udenafil-induced upregulation of IL-4, not IL12B, expression in ASCs. Three independent experiments were carried out per every experiment. *p<0.05; ***p<0.001. All error bars indicate SEM.
Fig. 7. Summary of hair promoting effect by udenafil. Udenafil upregulates the expression of IL-4 and IL12B via ERK or NFκB pathways, and releases two cytokines from ASC to promote hair growth.
References
  1. Ackermann, LW, and Denning, GM (2004). Nuclear factor-kappaB contributes to interleukin-4- and interferon-dependent polymeric immunoglobulin receptor expression in human intestinal epithelial cells. Immunology. 111, 75-85.
    Pubmed KoreaMed CrossRef
  2. Bao, K, and Reinhardt, RL (2015). The differential expression of IL-4 and IL-13 and its impact on type-2 immunity. Cytokine. 75, 25-37.
    Pubmed KoreaMed CrossRef
  3. Buhl, AE, Waldon, DJ, Kawabe, TT, and Holland, JM (1989). Minoxidil stimulates mouse vibrissae follicles in organ culture.. J Dermatol Sci. 92, 315-320.
    Pubmed
  4. Bunnell, BA, Estes, BT, Guilak, F, and Gimble, JM (2008). Differentiation of adipose stem cells.. Methods Mol Biol. 456, 155-171.
    Pubmed CrossRef
  5. Celik, SD, and Ates, O (2018). Genetic analysis of interleukin 18 gene polymorphisms in alopecia areata.. J. Clin. Lab. Anal.. 32, e22386.
    Pubmed CrossRef
  6. Choi, N, Shin, S, Song, SU, and Sung, JH (2018). Minoxidil promotes hair growth through stimulation of growth factor release from adipose-derived stem cells.. Int J Mol Sci. 19, E691.
    Pubmed KoreaMed CrossRef
  7. Dhayade, S, Kaesler, S, Sinnberg, T, Dobrowinski, H, Peters, S, Naumann, U, Liu, H, Hunger, RE, Thunemann, M, Biedermann, T, Schittek, B, Simon, HU, Feil, S, and Feil, R (2016). Sildenafil Potentiates a cGMP-Dependent Pathway to Promote Melanoma Growth.. Cell Rep. 14, 2599-2610.
    Pubmed CrossRef
  8. Festa, E, Fretz, J, Berry, R, Schmidt, B, Rodeheffer, M, Horowitz, M, and Horsley, V (2011). Adipocyte lineage cells contribute to the skin stem cell niche to drive hair cycling. Cell. 146, 761-771.
    Pubmed KoreaMed CrossRef
  9. Gopal, VK, Francis, SH, and Corbin, JD (2001). Allosteric sites of phosphodiesterase-5 (PDE5). A potential role in negative feedback regulation of cGMP signaling in corpus cavernosum.. Eur J Biochem. 268, 3304-3312.
    Pubmed CrossRef
  10. Gregoriou, S, Papafragkaki, D, Kontochristopoulos, G, Rallis, E, Kalogeromitros, D, and Rigopoulos, D (2010). Cytokines and other mediators in alopecia areata.. Mediators Inflamm. 2010, 928030.
    Pubmed KoreaMed CrossRef
  11. Groves, RW, Williams, IR, Sarkar, S, Nakamura, K, and Kupper, TS (1994). Analysis of epidermal IL-1 family members in vivo using transgenic mouse models.. J Investig Dermatol. 102, 556.
  12. Han, JH, Kwon, OS, Chung, JH, Cho, KH, Eun, HC, and Kim, KH (2004). Effect of minoxidil on proliferation and apoptosis in dermal papilla cells of human hair follicle.. J Dermatol Sci. 34, 91-98.
    Pubmed CrossRef
  13. Headington, JT (1987). Hair follicle biology and topical minoxidil: possible mechanisms of action. Dermatologica. 175, 19-22.
    Pubmed CrossRef
  14. Hoffmann, RT (1999). The potential role of cytokines and T cells in alopecia areata.. J Investig Dermatol Symp Proc. 4, 235-238.
    CrossRef
  15. Hoffmann, R, and Happle, R (1995). Does interleukin-1 induce hair loss?. Dermatology. 191, 273-275.
    Pubmed CrossRef
  16. Hoke, NN, Salloum, FN, Kass, DA, Das, A, and Kukreja, RC (2012). Preconditioning by phosphodiesterase-5 inhibition improves therapeutic efficacy of adipose-derived stem cells following myocardial infarction in mice. Stem Cells. 30, 326-335.
    Pubmed CrossRef
  17. Jeong, HH, Piao, S, Ha, JN, Kim, IG, Oh, SH, Lee, JH, Cho, HJ, Hong, SH, Kim, SW, and Lee, JY (2013a). Combined therapeutic effect of udenafil and adipose-derived stem cell (ADSC)/brain-derived neurotrophic factor (BDNF)-membrane system in a rat model of cavernous nerve injury. Urology. 81, 1108.e7-1108.e14.
    Pubmed CrossRef
  18. Jeong, YM, Sung, YK, Kim, WK, Kim, JH, Kwack, MH, Yoon, I, Kim, DD, and Sung, JH (2013b). Ultraviolet B preconditioning enhances the hair growth-promoting effects of adipose-derived stem cells via generation of reactive oxygen species.. Stem Cells Dev. 22, 158-168.
    Pubmed KoreaMed CrossRef
  19. Jin, SE, and Sung, JH (2016). Hair regeneration using adipose-derived stem cells.. Histol Histopathol. 31, 249-256.
    Pubmed CrossRef
  20. Kang, KK, Choi, SM, Ahn, GJ, Kwon, JW, and Kim, WB (2004). The effect of DA-8159 oncorpus cavernosal smooth muscle relaxation and penile erection in diabetic rabbits.. Urol Res. 32, 107-111.
    Pubmed CrossRef
  21. Kim, JH, Kim, WK, Sung, YK, Kwack, MH, Song, SY, Choi, JS, Park, SG, Yi, T, Lee, HJ, Kim, DD, Seo, HM, Song, SU, and Sung, JH (2014a). The molecular mechanism underlying the proliferating and preconditioning effect of vitamin C on adipose-derived stem cells.. Stem Cells Dev. 23, 1364-1376.
    Pubmed KoreaMed CrossRef
  22. Kim, JH, Park, SG, Kim, WK, Song, SU, and Sung, JH (2015). Functional regulation of adipose-derived stem cells by PDGFD. Stem Cells. 33, 542-556.
    Pubmed CrossRef
  23. Kim, JH, Park, SH, Park, SG, Choi, JS, Xia, Y, and Sung, JH (2011). The pivotal role of reactive oxygen species generation in the hypoxia-induced stimulation of adipose-derived stem cells.. Stem Cells Dev. 20, 1753-1761.
    Pubmed KoreaMed CrossRef
  24. Kim, WS, Han, L, Hwang, SJ, and Sung, JH (2014b). An update on niche composition, signaling and functional regulation of adipose-derived stem cell.. Expert Opin Biol Ther. 14, 1091-1102.
    Pubmed CrossRef
  25. Kim, WS, Park, BS, Sung, JH, Yang, JM, Park, SB, Kwak, SJ, and Park, JS (2007). Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts.. J Dermatol Sci. 48, 15-24.
    Pubmed CrossRef
  26. Kwack, MH, Kang, BM, Kim, MK, Kim, JC, and Sung, YK (2011). Minoxidil activates beta-catenin pathway in human dermal papilla cells: a possible explanation for its anagen prolongation effect.. J Dermatol Sci. 62, 154-159.
    Pubmed CrossRef
  27. Lee, CH, Kim, HS, Goo, MJ, Kang, KK, Ahn, BO, Kim, SH, and Yang, DY (2011). Chronic administration of udenafil, a selective phosphodiesterase type 5 inhibitor, promotes erectile function recovery in an animal model of bilateral cavernous nerve crush injury.. J Sex Med. 8, 1330-1340.
    Pubmed CrossRef
  28. Li, M, Marubayashi, A, Nakaya, Y, Fukui, K, and Arase, S (2001). Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.. J Invest Dermatol. 117, 1594-1600.
    Pubmed CrossRef
  29. Mandt, N, Geilen, CC, Wrobel, A, Gelber, A, Kamp, H, Orfanos, CE, and Blume-Peytavi, U (2002). Interleukin-4 induces apoptosis in cultured human follicular keratinocytes, but not in dermal papilla cells.. Eur J Dermatol. 12, 432-438.
    Pubmed
  30. Michelet, JF, Commo, S, Billoni, N, Mahe, YF, and Bernard, BA (1997). Activation of cytoprotective prostaglandin synthase-1 by minoxidil as a possible explanation for its hair growth-stimulating effect.. J Invest Dermatol. 108, 205-209.
    Pubmed CrossRef
  31. Otomo, S (2002). Hair growth effect of minoxidil. Nihon Yakurigaku Zasshi. 119, 167-74.
    Pubmed CrossRef
  32. Paick, JS, Kim, SW, Park, YK, Hyun, JS, Park, NC, Lee, SW, Park, K, Moon, KH, and Chung, WS (2009). The efficacy and safety of udenafil [Zydena] for the treatment of erectile dysfunction in hypertensive men taking concomitant antihypertensive agents.. J Sex Med. 6, 3166-3176.
    Pubmed CrossRef
  33. Perez-Meza, D, Ziering, C, Sforza, M, Krishnan, G, Ball, E, and Daniels, E (2017). Hair follicle growth by stromal vascular fraction-enhanced adipose transplantation in baldness. Stem Cells Cloning. 10, 1-10.
    Pubmed KoreaMed CrossRef
  34. Pyriochou, A, Zhou, Z, Koika, V, Petrou, C, Cordopatis, P, Sessa, WC, and Papapetropoulos, A (2007). The phosphodiesterase 5 inhibitor sildenafil stimulates angiogenesis through a protein kinase G/MAPK pathway.. J Cell Physiol. 211, 197-204.
    Pubmed CrossRef
  35. Ramot, Y, Marzani, B, Pinto, D, Sorbellini, E, and Rinaldi, F (2018). IL-17 inhibition: is it the long-awaited savior for alopecia areata?. Arch Dermatol Res. 310, 383-390.
    Pubmed CrossRef
  36. Redler, S, Albert, F, Brockschmidt, FF, Herold, C, Hanneken, S, Eigelshoven, S, Giehl, KA, Kruse, R, Lutz, G, Wolff, H, Blaumeiser, B, Böhm, M, Becker, T, Nöthen, MM, and Betz, RC (2012). Investigation of selected cytokine genes suggests that IL-2RA and the TNF/LTA locus are risk factors for severe alopecia areata.. Br J Dermatol. 167, 1360-1365.
    Pubmed CrossRef
  37. Sahara, M, Sata, M, Morita, T, Nakajima, T, Hirata, Y, and Nagai, R (2010). A phosphodiesterase-5 inhibitor vardenafil enhances angio-genesis through a protein kinase G-dependent hypoxia-inducible factor-1/vascular endothelial growth factor pathway.. Arterioscler Thromb Vasc Biol. 30, 1315-1324.
    Pubmed CrossRef
  38. Won, CH, Yoo, HG, Kwon, OS, Sung, MY, Kang, YJ, Chung, JH, Park, BS, Sung, JH, Kim, WS, and Kim, KH (2010). Hair growth promoting effects of adipose tissue-derived stem cells.. J Dermatol Sci. 57, 134-137.
    Pubmed CrossRef
  39. Yang, Y, Choi, H, Seon, M, Cho, D, and Bang, SI (2016). LL-37 stimulates the functions of adipose-derived stromal/stem cells via early growth response 1 and the MAPK pathway.. Stem Cell Res Ther. 7, 58.
    Pubmed KoreaMed CrossRef
  40. Yi, T, Kim, WK, Choi, JS, Song, SY, Han, J, Kim, JH, Kim, WS, Park, SG, Lee, HJ, Cho, YK, Hwang, SJ, Song, SU, and Sung, JH (2014). Isolation of adipose-derived stem cells by using a subfractionation culturing method.. Expert Opin Biol Ther. 14, 1551-1560.
    Pubmed CrossRef


This Article


Cited By Articles
  • CrossRef (0)

Funding Information
  • National Research Foundation of Korea
      10.13039/501100003725
      NRF-2016R1D1A1B03932050, NRF-2017R1A6A3A11035599

Services
Social Network Service

e-submission

Archives