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
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
We have identified stimulators that enhance the hair growth potential of ASCs
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
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.
Human ASCs were isolated via liposuction of subcutaneous fat, as described in a previous study (Kim
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.
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.
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.
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.
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).
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.
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
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 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).
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
Although ASCs secrete various growth factors that promotes hair growth (Won
When we investigated the hair growth effect of udenafilpretreated ASCs
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
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.
Previous studies have reported that the MAPK pathway regulates growth factor expression in ASCs, including VEGF-A and FGF2 (Kim
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.
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
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
Although ASC transplantation promotes hair growth
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
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).
The authors have no conflicts of interest to declare and have not received any payment for the preparation of this manuscript.
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.