Adenosine Promotes Human Hair Growth and Inhibits Catagen Transition In Vitro: Role of the Outer Root Sheath Keratinocytes
2019; Elsevier BV; Volume: 140; Issue: 5 Linguagem: Inglês
10.1016/j.jid.2019.08.456
ISSN1523-1747
AutoresErika Lisztes, Balázs I. Tóth, Marta Bertolini, Imre Szabó, Nóra Zákány, Attila Oláh, Attila Gábor Szöllősi, Ralf Paus, Tamás Bı́ró,
Tópico(s)Dermatologic Treatments and Research
ResumoAdenosine is a locally produced mediator exerting several cytoprotective effects via G-protein coupled cell membrane adenosine receptors (ARs) (Linden, 2005Linden J. Adenosine in tissue protection and tissue regeneration.Mol Pharmacol. 2005; 67: 1385-1387Crossref PubMed Scopus (207) Google Scholar). In the skin, adenosine can influence several (patho)physiological processes, such as wound healing, development of scleroderma, cutaneous inflammation, allergic reactions, or barrier formation (Andrés et al., 2017Andrés R.M. Terencio M.C. Arasa J. Payá M. Valcuende-Cavero F. Navalón P. et al.Adenosine A2A and A2B receptors differentially modulate keratinocyte proliferation: possible deregulation in psoriatic epidermis.J Invest Dermatol. 2017; 137: 123-131Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, Burnstock et al., 2012Burnstock G. Knight G.E. Greig A.V.H. Purinergic signaling in healthy and diseased skin.J Invest Dermatol. 2012; 132: 526-546Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, Silva-Vilches et al., 2019Silva-Vilches C. Ring S. Schrader J. Clausen B.E. Probst H.C. Melchior F. et al.Production of extracellular adenosine by CD73+ dendritic cells is crucial for induction of tolerance in contact hypersensitivity reactions.J Invest Dermatol. 2019; 139: 541-551Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). A beneficial effect of adenosine on hair growth has already been reported in clinical studies; topical adenosine treatment was shown to alleviate the symptoms of alopecia by increasing hair thickness and promoting anagen hair growth (Iwabuchi et al., 2016Iwabuchi T. Ideta R. Ehama R. Yamanishi H. Iino M. Nakazawa Y. et al.Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia.J Dermatol. 2016; 43: 567-570Crossref PubMed Scopus (9) Google Scholar, Oura et al., 2008Oura H. Iino M. Nakazawa Y. Tajima M. Ideta R. Nakaya Y. et al.Adenosine increases anagen hair growth and thick hairs in Japanese women with female pattern hair loss: a pilot, double-blind, randomized, placebo-controlled trial.J Dermatol. 2008; 35: 763-767Crossref PubMed Scopus (24) Google Scholar, Watanabe et al., 2015Watanabe Y. Nagashima T. Hanzawa N. Ishino A. Nakazawa Y. Ogo M. et al.Topical adenosine increases thick hair ratio in Japanese men with androgenetic alopecia.Int J Cosmet Sci. 2015; 37: 579-587Crossref PubMed Scopus (11) Google Scholar), whereas adenosine also prolonged the anagen phase of mouse vibrissae cultures (Hwang et al., 2012Hwang K.A. Hwang Y.L. Lee M.H. Kim N.R. Roh S.S. Lee Y. et al.Adenosine stimulates growth of dermal papilla and lengthens the anagen phase by increasing the cysteine level via fibroblast growth factors 2 and 7 in an organ culture of mouse vibrissae hair follicles.Int J Mol Med. 2012; 29: 195-201PubMed Google Scholar). In parallel experiments, it upregulated the expression of fibroblast growth factor 7 via activating A2B AR and stimulated the transcription of other growth factors in human dermal papilla cell cultures (Hwang et al., 2012Hwang K.A. Hwang Y.L. Lee M.H. Kim N.R. Roh S.S. Lee Y. et al.Adenosine stimulates growth of dermal papilla and lengthens the anagen phase by increasing the cysteine level via fibroblast growth factors 2 and 7 in an organ culture of mouse vibrissae hair follicles.Int J Mol Med. 2012; 29: 195-201PubMed Google Scholar, Iino et al., 2007Iino M. Ehama R. Nakazawa Y. Iwabuchi T. Ogo M. Tajima M. et al.Adenosine stimulates fibroblast growth factor-7 gene expression via adenosine A2b receptor signaling in dermal papilla cells.J Invest Dermatol. 2007; 127: 1318-1325Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). However, the influence of adenosine on the mechanisms of hair growth is not completely understood, and the role of ARs expressed in the different compartments of hair follicles (HFs) is not known in detail. Therefore, we addressed the role of adenosine and ARs in hair cycle control studying isolated human HFs and primary outer root sheath (ORS) keratinocytes in vitro as described in the Supplementary Materials and Methods. Human HFs were isolated from human skin samples obtained from healthy individuals undergoing neurosurgery with written informed consent, adhering to Helsinki guidelines and after obtaining the permission of the Institutional Research Ethics Committee and the Government Office for Hajdú-Bihar County (protocol No.: DE OEC RKEB/IKEB 3724-2012; document IDs: IX-R-052/01396-2/2012, IF-12817/2015, IF-1647/2016, IF-778-5/2017). First, we treated microdissected human HFs with adenosine administered in the culturing medium for several days and measured the length of the HFs on the first, third, and fifth days. We found that hair shaft elongation was stimulated in the presence of 50 or 100 μM adenosine (Figure 1a). These concentrations of adenosine increased cellular proliferation in HFs, especially in the matrix keratinocytes of the hair bulb as indicated by the increased number of cells positive for the proliferation-associated antigen Ki-67 (Supplementary Figure S1a and b). In good accordance with the previous results, adenosine slightly shifted the hair cycle by prolonging the duration of the anagen phase and inhibiting catagen entry; the ratio of HFs in the anagen stage was higher when 100 μM adenosine was added to the culture medium for 6 days, as assessed by hair cycle staging based on the histomorphometric evaluation of the cultured HFs (Supplementary Figure S1c and d). Next, we wanted to further challenge the anagen-promoting effect of adenosine by the co-administration of the catagen inducer transforming growth factor β2 (TGF-β2) (Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar). As expected, TGF-β2 significantly inhibited hair growth, decreased proliferation, and induced apoptosis of hair matrix keratinocytes in cultured HFs (Figure 1b–d). TGF-β2 also resulted in a striking catagen transition of the hair cycle, and it practically abolished the anagen stage from the cultures in 6 days (Figure 1e and f, Supplementary Figure S2a and b). All these effects of TGF-β2 were mainly prevented by supporting the culture medium with 100 μM adenosine. Importantly, this blockade of TGF-β2’s effect by adenosine was abrogated by the co-application of CGS15943, a pan-antagonist of ARs. Therefore, we investigated the presence of ARs in human HFs. We isolated total mRNA from anagen human HFs from three donors and determined the expression of ARs A1, A2A, A2B, and A3 by quantitative PCR following reverse transcription. We found that the transcripts of all four investigated receptors are expressed in HFs (Figure 2a), among which A2B was found to be the dominant isoform; its expression was at least 1 magnitude higher than any other isoform in the tested donors. We studied the localization of AR proteins within the HF by applying immunofluorescent labeling on frozen sections of isolated human anagen HFs (Figure 2b) using specific antibodies against ARs (Supplementary Figure S3). A1 showed a diffuse expression overall in the matrix and ORS keratinocytes, whereas A2A and A2B isoforms were expressed in the ORS and some cells in the dermal papilla, in line with the previous results of Iino et al., 2007Iino M. Ehama R. Nakazawa Y. Iwabuchi T. Ogo M. Tajima M. et al.Adenosine stimulates fibroblast growth factor-7 gene expression via adenosine A2b receptor signaling in dermal papilla cells.J Invest Dermatol. 2007; 127: 1318-1325Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, who investigated the expression of A2B in human scalp specimens and dermal papilla cells. The surrounding connective tissue sheath was also positive for A2B, and some A2A was also detected in the inner root sheath. A3 was detected primarily in matrix keratinocytes above the Auber’s line, and a weak signal was observed in the ORS. We isolated keratinocytes from the ORS because immunofluorescence detected all ARs in that compartment and further studied the expression of ARs in the isolated cells. Quantitative PCR and immunofluorescent staining confirmed the results obtained in HF cultures; even in monolayer cultures, ORS keratinocytes expressed all four ARs (Figure 2c and d). Similar to HFs, the expression of the A2B transcripts was found to be higher than the expression of the other isoforms (Figure 2c). In our final experiments, we further dissected the potential mechanisms of adenosine on hair growth and the hair cycle. Because the ORS is well-known to synthesize both positive and negative paracrine regulators of hair growth and the hair cycle (e.g., TGF-β2, insulin-like growth factor 1, or stem cell factor/c-kit ligand [Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Griger Z. Tóth B.I. et al.A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Google Scholar, Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar, Paus et al., 2014Paus R. Langan E.A. Vidali S. Ramot Y. Andersen B. Neuroendocrinology of the hair follicle: principles and clinical perspectives.Trends Mol Med. 2014; 20: 559-570Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Soma et al., 2002Soma T. Tsuji Y. Hibino T. Involvement of transforming growth factor-β2 in Catagen Induction during the Human Hair Cycle.J Invest Dermatol. 2002; 118: 993-997Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, Stenn and Paus, 2001Stenn K.S. Paus R. Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (950) Google Scholar, Szabó et al., 2019Szabó I.L. Herczeg-Lisztes E. Szegedi A. Nemes B. Paus R. Bíró T. et al.TRPV4 is expressed in human hair follicles and inhibits hair growth in vitro.J Invest Dermatol. 2019; 139: 1385-1388Abstract Full Text Full Text PDF PubMed Google Scholar]), we studied the effect of adenosine on the expression of selected, well-established hair cycle regulators in ORS keratinocytes isolated from three donors. Detecting the specific mRNA transcripts, we found that adenosine downregulated the expression of the catagen-inducing mediators TGF-β2 and epidermal growth factor. In contrast, expression of the anagen and pigmentation promoting mediator stem cell factor was upregulated by adenosine treatment, as well as the expression of insulin-like growth factor 1 receptor (Figure 2e). These alterations in gene expression induced by adenosine were observable in the samples of each donor. Importantly, the effect of adenosine was blocked by both the general AR receptor antagonist CGS15943 and MRS1754, an A2B selective inhibitor, in the case of each donor, although the magnitude of the responses was variable among donors tested. These data demonstrated convincingly that adenosine can control the hair cycle via ARs expressed in human HFs. Moreover, our results also suggest that adenosine can regulate complex intercellular signaling pathways in HFs acting on ARs (especially A2B) expressed by ORS keratinocytes beyond previously described dermal papilla cells. Our findings describe the growth and anagen-promoting effect of adenosine via ARs in human HFs and identify especially A2B expressed in ORS keratinocytes as a promising pharmacological target to influence various hair growth disorders, such as various forms of alopecia. Data sets related to this article are freely available upon request. Requests should be addressed to the corresponding author. Erika Lisztes: http://orcid.org/0000-0002-8517-6536 Balázs István Tóth: http://orcid.org/0000-0002-4103-4333 Marta Bertolini: http://orcid.org/0000-0002-5927-6998 Imre Lőrinc Szabó: http://orcid.org/0000-0002-9628-4372 Nóra Zákány: http://orcid.org/0000-0003-4239-6106 Attila Oláh: http://orcid.org/0000-0003-4122-5639 Attila Gábor Szöllősi: http://orcid.org/0000-0001-6046-8236 Ralf Paus: http://orcid.org/0000-0002-3492-9358 Tamás Bíró: http://orcid.org/0000-0002-3770-6221 MB is an employee of Monasterium Laboratory GmbH. RP is the founder and CEO of Monasterium Laboratory GmbH. TB and AO provide consultancy services to Phytecs Inc. (TB) and Botanix Pharmaceuticals Ltd. (AO). Botanix Pharmaceuticals Ltd., Phytecs Inc., Monasterium Laboratory GmbH, and the founding sponsors listed in the Acknowledgments section had no role in conceiving the study, designing the experiments, writing of the manuscript, or in the decision to publish it. The other authors state no conflict of interest. The presented work was supported by research grants of the National Research, Development and Innovation Office (K_120187, PD_121360, FK_125055, GINOP-2.3.2-15-2016-00050). The project has received funding from the EU’s Horizon 2020 research and innovation program under grant agreement No. 739593. AO’s work was supported by the New National Excellence Program of the Ministry of Human Capacities (ÚNKP-18-4-DE-247). BIT, AO, and AGSz are recipients of the János Bolyai research scholarship of the Hungarian Academy of Sciences. Conceptualization: EL, BIT, AO, RP, TB; Data Curation: EL, BIT, MB, ILSz, NZ, AO, AGSz; Formal Analysis: EL, BIT, TB; Funding Acquisition: BT; Writing - Original Draft Preparation: EL, BIT, BT; Writing - Review and Editing: RP. Human anagen VI HFs were isolated from the skin of male donors and maintained as described previously (Bodó et al., 2009Bodó E. Kromminga A. Bíró T. Borbíró I. Gáspár E. Zmijewski M.A. et al.Human female hair follicles are a direct, nonclassical target for thyroid-stimulating hormone.J Invest Dermatol. 2009; 129: 1126-1139Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 2005; Borbíró et al., 2011Borbíró I. Lisztes E. Tóth B.I. Czifra G. Oláh A. Szöllosi A.G. et al.Activation of transient receptor potential vanilloid-3 inhibits human hair growth.J Invest Dermatol. 2011; 131: 1605-1614Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Oláh et al., 2016Oláh A. Gherardini J. Bertolini M. Chéret J. Ponce L. Kloepper J. et al.The thyroid hormone analogue KB2115 (eprotirome) prolongs human hair growth (Anagen) ex vivo.J Invest Dermatol. 2016; 136: 1711-1714Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Ramot et al., 2010Ramot Y. Bíró T. Tiede S. Tóth B.I. Langan E.A. Sugawara K. et al.Prolactin--a novel neuroendocrine regulator of human keratin expression in situ.FASEB J. 2010; 24: 1768-1779Crossref PubMed Scopus (55) Google Scholar, Szabó et al., 2019Szabó I.L. Herczeg-Lisztes E. Szegedi A. Nemes B. Paus R. Bíró T. et al.TRPV4 is expressed in human hair follicles and inhibits hair growth in vitro.J Invest Dermatol. 2019; 139: 1385-1388Abstract Full Text Full Text PDF PubMed Google Scholar, Telek et al., 2007Telek A. Bíró T. Bodó E. Tóth B.I. Borbíró I. Kunos G. et al.Inhibition of human hair follicle growth by endo- and exocannabinoids.FASEB J. 2007; 21: 3534-3541Crossref PubMed Scopus (72) Google Scholar). Briefly, isolated HFs were collected and maintained in Williams’ E medium (Life Technologies, Carlsbad, CA) supplemented with 2 mM L-glutamine (Life Technologies), 10 ng/ml hydrocortisone, 10 μg/ml insulin, and antibiotics (all from Sigma-Aldrich, St. Louis, MO). Culture medium was changed every other day, whereas treatment with various compounds was performed daily. For immunofluorescent staining and histomorphometry, HFs were frozen at −80 °C and further processed after 6 days in culture. Plucked human scalp HFs of several male volunteers were digested using trypsin to obtain ORS keratinocytes (Ramot et al., 2018Ramot Y. Alam M. Oláh A. Bíró T. Ponce L. Chéret J. et al.Peroxisome proliferator-activated receptor-γ-mediated signaling regulates mitochondrial energy metabolism in human hair follicle epithelium.J Invest Dermatol. 2018; 138: 1656-1659Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Similarly, human dermal fibroblasts were obtained from de-epidermized dermis of human skin samples using enzymatic digestion. ORS keratinocyte cultures were kept on a feeder layer of nonproliferating human dermal fibroblasts treated with mitomycin C (Sigma-Aldrich) in a 1:3 mixture of supplemented Ham’s F12 and DMEM (both from Life Technologies) supplemented with 10% Fetal Clone II (HyClone, Logan, UT), 0.1 nM cholera toxin, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 2.43 μg/ml adenine, 2 nM triiodothyronine, 10 ng/ml epidermal growth factor, 1 mM ascorbyl-2-phosphate, and antibiotics (all from Sigma-Aldrich) as described previously (Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Griger Z. Tóth B.I. et al.A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Google Scholar, Borbíró et al., 2011Borbíró I. Lisztes E. Tóth B.I. Czifra G. Oláh A. Szöllosi A.G. et al.Activation of transient receptor potential vanilloid-3 inhibits human hair growth.J Invest Dermatol. 2011; 131: 1605-1614Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Ramot et al., 2018Ramot Y. Alam M. Oláh A. Bíró T. Ponce L. Chéret J. et al.Peroxisome proliferator-activated receptor-γ-mediated signaling regulates mitochondrial energy metabolism in human hair follicle epithelium.J Invest Dermatol. 2018; 138: 1656-1659Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, Szabó et al., 2019Szabó I.L. Herczeg-Lisztes E. Szegedi A. Nemes B. Paus R. Bíró T. et al.TRPV4 is expressed in human hair follicles and inhibits hair growth in vitro.J Invest Dermatol. 2019; 139: 1385-1388Abstract Full Text Full Text PDF PubMed Google Scholar, Telek et al., 2007Telek A. Bíró T. Bodó E. Tóth B.I. Borbíró I. Kunos G. et al.Inhibition of human hair follicle growth by endo- and exocannabinoids.FASEB J. 2007; 21: 3534-3541Crossref PubMed Scopus (72) Google Scholar). Before experiments, ORS keratinocytes were harvested and replated without a human dermal fibroblast feeder layer in 6-well plates (200,000 cells/well) that were previously coated with 1% collagen (Sigma-Aldrich) and kept in serum-free medium for 24 hours before treating them with different compounds. Three hours after treatment, cells were harvested using TRIzol (Life Technologies) and quantitative real-time reverse transcriptase–PCR was performed as described later. Length measurements on individual cultured HFs were performed using a light microscope with an eyepiece measuring graticule. Elongation was calculated for each hair follicle separately, by subtracting the length measured on day 0 from the value of the relevant day. Total RNA was isolated using TRIzol reagent (Life Technologies) and digested with recombinant RNase-free DNase-1 (Life Technologies) according to the manufacturer’s protocol. After isolation, 1 μg of total RNA was reverse-transcribed into cDNA using the High Capacity cDNA kit (Life Technologies) following the manufacturer’s instructions. Quantitative real-time PCR was performed on a Stratagene Mx3005p sequence detection system (Agilent Technologies, Santa Clara, CA) by using 5′ nuclease assay. PCR amplification was performed using specific TaqMan primers and probes as follows: adenosine A1 receptor (assay ID: Hs00379752_m1), adenosine A2A receptor (assay ID: Hs00169123_m1), adenosine A2B receptor (assay ID: Hs00386497_m1), adenosine A3 receptor (assay ID: Hs00252933_m1), transforming growth factor β2 (TGF-β2, assay ID: Hs00234244_m1), epidermal growth factor (assay ID: Hs01099999_m1), stem cell factor/KIT-ligand (assay ID: Hs00241497_m1), and insulin-like growth factor 1 receptor (assay ID: Hs00609566_m1) using the TaqMan Gene Expression Master Mix Protocol (Life Technologies). As internal control, transcripts of cyclophilin A (assay ID: Hs99999904_m1) were determined. The amount of the aforementioned transcripts was normalized to the expression of the internal control gene using the ΔCt method. Briefly, the threshold cycle (Ct) value of the target gene was subtracted from the average Ct value of the control gene resulting in the ΔCt value. ΔCt was then used as a power of two, which results in the relative expression of a given target gene compared with the control (i.e., 2ΔCt). All experiments were performed in triplicate. To detect the four types of ARs on isolated HFs and ORS keratinocytes, we performed indirect fluorescent immunolabeling. Cryosections of isolated HFs fixed with ice-cold ethanol:acetic acid (2:1) or acetone-fixed ORS keratinocytes grown on coverslips were first incubated with different primary rabbit antibodies (1:100 in DCS antibody diluent [DCS Innovative Diagnostik-Systeme, Hamburg, Germany] overnight, 4 °C) against A1, A2A (Abcam, Cambridge, United Kingdom, cat. numbers: ab124780 and ab3461, respectively), A2B, and A3 (Alomone Labs, Jerusalem, Israel, cat. numbers: AAR-003 and AAR-004, respectively) receptors. Sections and coverslips were then washed with phosphate buffered saline, followed by incubation with Alexa Fluor 488 dye-conjugated goat anti-rabbit IgG (Life Technologies) (1:500 in DCS antibody diluent, 45 minutes) at room temperature according to standard procedures. Nuclei were counterstained with DAPI (Life Technologies, 1 μg/ml in distilled water, 5 minutes), and sections were mounted with Fluoromount-G aqueous medium (Southern Biotech, Birmingham, AL). Images were acquired using an Eclipse E600 fluorescent microscope (Nikon, Tokyo, Japan). To verify the specificity of the antibodies used, paraffin-embedded routine histology sections from tissues known to express different ARs were stained as positive controls. Human cerebral cortex served as positive control for A1 and A2A (Latini et al., 1996Latini S. Pazzagli M. Pepeu G. Pedata F. A2 adenosine receptors: their presence and neuromodulatory role in the central nervous system.Gen Pharmacol. 1996; 27: 925-933Crossref PubMed Scopus (78) Google Scholar, Luan et al., 2017Luan G. Wang X. Gao Q. Guan Y. Wang J. Deng J. et al.Upregulation of neuronal adenosine A1 receptor in human Rasmussen encephalitis.J Neuropathol Exp Neurol. 2017; 76: 720-731Crossref PubMed Scopus (13) Google Scholar, Svenningsson et al., 1997Svenningsson P. Hall H. Sedvall G. Fredholm B.B. Distribution of adenosine receptors in the postmortem human brain: an extended autoradiographic study.Synapse. 1997; 27: 322-335Crossref PubMed Scopus (208) Google Scholar), human kidney for A2B (Zhang et al., 2013Zhang W. Zhang Y. Wang W. Dai Y. Ning C. Luo R. et al.Elevated ecto-5’-nucleotidase-mediated increased renal adenosine signaling via A2B adenosine receptor contributes to chronic hypertension.Circ Res. 2013; 112: 1466-1478Crossref PubMed Scopus (64) Google Scholar), and human cerebellum for A3 (Haeusler et al., 2015Haeusler D. Grassinger L. Fuchshuber F. Hörleinsberger W.J. Höftberger R. Leisser I. et al.Hide and seek: a comparative autoradiographic in vitro investigation of the adenosine A3 receptor.Eur J Nucl Med Mol Imaging. 2015; 42: 928-939Crossref PubMed Scopus (14) Google Scholar). Following deparaffination and antigen retrieval (in citrate buffer, pH 6.0, at 750 W in microwave oven for 15 minutes), sections were incubated with the previously described primary rabbit antibodies against human ARs, then stained with horseradish peroxidase–conjugated anti-rabbit IgG (1:500) (Bio-Rad, Hercules, CA). Immunoreactions were visualized using DAB substrate kit (Vector Labs, Burlingame, CA) and the sections were counterstained by hematoxylin (Sigma-Aldrich). For all immunostainings, the respective primary antibodies were omitted as negative controls. To simultaneously detect proliferating and apoptotic cells in the HFs, Ki-67 immunolabeling and TUNEL, respectively, were performed in a double-staining protocol (Bodó et al., 2005Bodó E. Bíró T. Telek A. Czifra G. Griger Z. Tóth B.I. et al.A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control.Am J Pathol. 2005; 166: 985-998Abstract Full Text Full Text PDF PubMed Google Scholar, Borbíró et al., 2011Borbíró I. Lisztes E. Tóth B.I. Czifra G. Oláh A. Szöllosi A.G. et al.Activation of transient receptor potential vanilloid-3 inhibits human hair growth.J Invest Dermatol. 2011; 131: 1605-1614Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar, Purba et al., 2016Purba T.S. Brunken L. Hawkshaw N.J. Peake M. Hardman J. Paus R. A primer for studying cell cycle dynamics of the human hair follicle.Exp Dermatol. 2016; 25: 663-668Crossref PubMed Scopus (32) Google Scholar; Szabó et al., 2019; Telek et al., 2007Telek A. Bíró T. Bodó E. Tóth B.I. Borbíró I. Kunos G. et al.Inhibition of human hair follicle growth by endo- and exocannabinoids.FASEB J. 2007; 21: 3534-3541Crossref PubMed Scopus (72) Google Scholar). Cryosections were fixed in formalin/ethanol/acetic acid and labeled with a digoxigenin-deoxyUTP (ApopTag Fluorescein In Situ Apoptosis detection kit; Millipore, Burlington, MA) in the presence of terminal deoxynucleotidyl transferase (60 minutes, 4 °C) according to the manufacturers protocol, followed by overnight incubation with a mouse anti–Ki-67 antiserum (1:20, Dako, Carpinteria, CA) at 4 °C. TUNEL+ cells were visualized by an antidigoxigenin FITC-conjugated antibody (ApopTag kit), whereas Ki-67 was detected by an Alexa Fluor 568 dye-conjugated secondary antibody (Life Technologies, 1:500 at 4 °C for 45 minutes). Negative control stainings were performed by omitting terminal deoxynucleotidyl transferase and the Ki-67 antibody (data not shown). Cells positive for Ki-67 or TUNEL were counted per hair bulb (under the cross-sectional line perpendicular for the longitudinal axis of the hair shaft and tangential to the peak of the dermal papilla) and were normalized to the number of nuclei (DAPI+). Cryosections (6 μm) of cultured HFs were fixed in acetone, air-dried, and processed for routine histology. Hematoxylin and eosin (Sigma-Aldrich) staining was used for studying HF morphology, and hair cycle stage (anagen and different stages of catagen) of each HF was assessed according to defined morphological criteria (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N. Paus R. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (100) Google Scholar, Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar). Additionally, number of DAPI+ cells in a standardized area of the dermal papilla stalk was counted on Ki-67 and TUNEL double-labeled sections to further characterize hair cycle quantitatively (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N. Paus R. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture.Exp Dermatol. 2010; 19: 305-312Crossref PubMed Scopus (100) Google Scholar). If not mentioned otherwise, values are presented as mean ± SEM in every group. To compare the mean values of multiple groups, statistical analysis was subsequently performed by one-way analysis of variance and Dunnett or Bonferroni post hoc tests, as appropriate. Significance was determined as compared with the control and/or compared with different treated samples as indicated. Differences in distribution of HFs among different hair cycle stages was compared pairwise by Fisher’s exact test. Origin 9.0 (OriginLab Corporation, Northampton MA) and IBM SPSS Statistics 23.0 (IBM Corporation. Armonk, NY) were used to plot the data and perform statistical analysis, respectively. Adenosine, CGS15943 (non-selective AR antagonist), MRS1754 (selective A2B antagonist), and TGF-β2 were obtained from Sigma-Aldrich. Data sets related to this article are freely available upon request. Requests should be addressed to the corresponding author. The favorable action of adenosine is supported by a growing body of evidence in HF biology, but the potential mechanism of action has not been resolved yet. To get deeper insight into the cellular and molecular mechanisms of how adenosine can enhance human hair growth, we studied the effect of adenosine in an in vitro model of human hair growth using microdissected and organ-cultured human HFs (Langan et al., 2015Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (56) Google Scholar, Philpott et al., 1994Philpott M.P. Sanders D. Westgate G.E. Kealey T. Human hair growth in vitro: a model for the study of hair follicle biology.J Dermatol Sci. 1994; 7: S55-S72Abstract Full Text PDF PubMed Scopus (115) Google Scholar). In good accordance with the previous clinical findings, we quantitatively measured that adenosine enhanced the hair shaft elongation in human HF cultures isolated from Caucasian male subjects in vitro. As a potential underlying mechanism, we found increased intrafollicular proliferation and also observed that the ratio of HFs in catagen stage was decreased and more HFs showed morphological signs characteristic for the growing anagen phase in the adenosine-treated cultu
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