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The Phytocannabinoid (–)-Cannabidiol Operates as a Complex, Differential Modulator of Human Hair Growth: Anti-Inflammatory Submicromolar versus Hair Growth Inhibitory Micromolar Effects

2019; Elsevier BV; Volume: 140; Issue: 2 Linguagem: Inglês

10.1016/j.jid.2019.07.690

ISSN

1523-1747

Autores

Imre Szabó, Erika Lisztes, Gabriella Béke, Kinga Tóth, Ralf Paus, Attila Oláh, Tamás Bı́ró,

Tópico(s)

Toxin Mechanisms and Immunotoxins

Resumo

Most cases of excessive hair loss and unwanted hair growth (e.g., effluvium, alopecia, hirsutism, hypertrichosis) result in part from major disturbances in the cyclic transformation of hair follicles (HFs), namely in their switch from active growth and pigmented hair shaft production (anagen) to apoptosis-driven HF involution (catagen) (Oh et al., 2016Oh J.W. Kloepper J. Langan E.A. Kim Y. Yeo J. Kim M.J. et al.A guide to studying human hair follicle cycling in vivo.J Invest Dermatol. 2016; 136: 34-44Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, Paus and Cotsarelis, 1999Paus R. Cotsarelis G. The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (883) Google Scholar). It is now clear that this switch is profoundly influenced by complex neuroendocrine pathways, which still await systematic therapeutic targeting (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 (72) Google Scholar). In this context, the endocannabinoid system is of special interest (Maccarrone et al., 2015Maccarrone M. Bab I. Bíró T. Cabral G.A. Dey S.K. Di Marzo V. et al.Endocannabinoid signaling at the periphery: 50 years after THC.Trends Pharmacol Sci. 2015; 36: 277-296Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, Oláh and Bíró, 2017Oláh A. Bíró T. Targeting cutaneous Cannabinoid Signaling in Inflammation - A "High"-way to Heal?.EBioMedicine. 2017; 16: 3-5Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, Tóth et al., 2019Tóth K.F. Ádám D. Bíró T. Oláh A. Cannabinoid Signaling in the Skin: therapeutic Potential of the "C(ut)annabinoid" System.Molecules. 2019; 24: E918Crossref PubMed Scopus (62) Google Scholar). Specifically, activation of CB1 receptors by the endocannabinoid anandamide, and the plant-derived (–)-Δ9-trans-tetrahydrocannabinol, promotes premature catagen entry in human scalp HFs ex vivo (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 (63) Google Scholar). However, 2-arachidonoylglycerol, another prototypic endocannabinoid, does not modulate HF growth ex vivo, suggesting functional heterogeneity between different cannabinoids in terms of human hair growth modulation (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 (63) Google Scholar). This motivated us to also explore how another phytocannabinoid, (–)-cannabidiol (CBD), impacts on human hair growth. This major nonpsychotropic phytocannabinoid does not activate CB1 receptors, but may context-dependently signal via modulating several other targets (e.g., GPR55, 5-HT1a, or μ-opioid receptors) (Pertwee, 2008Pertwee R.G. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: delta 9-tetrahydrocannabinol, cannabidiol and delta 9-tetrahydrocannabivarin.Br J Pharmacol. 2008; 153: 199-215Crossref PubMed Scopus (968) Google Scholar). Moreover, it is used in clinical practice in multiple sclerosis and certain epilepsies, and is currently under intense investigation for various uses in clinical medicine, ranging from neuropsychiatric disorders to acne (ID at ClinicalTrials.gov: NCT03573518) (Armstrong et al., 2015Armstrong J.L. Hill D.S. McKee C.S. Hernandez-Tiedra S. Lorente M. Lopez-Valero I. et al.Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.J Invest Dermatol. 2015; 135: 1629-1637Abstract Full Text Full Text PDF PubMed Google Scholar, Chelliah et al., 2018Chelliah M.P. Zinn Z. Khuu P. Teng J.M.C. Self-initiated use of topical cannabidiol oil for epidermolysis bullosa.Pediatr Dermatol. 2018; 35: e224-e227Crossref PubMed Scopus (35) Google Scholar, Crippa et al., 2018Crippa J.A. Guimarães F.S. Campos A.C. Zuardi A.W. Translational investigation of the therapeutic potential of cannabidiol (CBD): toward a new age.Front Immunol. 2018; 9: 2009Crossref PubMed Scopus (85) Google Scholar, Esposito et al., 2013Esposito G. Filippis D.D. Cirillo C. Iuvone T. Capoccia E. Scuderi C. et al.Cannabidiol in inflammatory bowel diseases: a brief overview.Phytother Res. 2013; 27: 633-636Crossref PubMed Scopus (47) Google Scholar, Pisanti et al., 2017Pisanti S. Malfitano A.M. Ciaglia E. Lamberti A. Ranieri R. Cuomo G. et al.Cannabidiol: state of the art and new challenges for therapeutic applications.Pharmacol Ther. 2017; 175: 133-150Crossref PubMed Scopus (199) Google Scholar). In fact, CBD exerts complex sebostatic and anti-inflammatory effects on the pilosebaceous unit by activating both transient receptor potential vanilloid-4 (TRPV4) ion channels and adenosine A2A receptors on sebocytes (Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (76) Google Scholar). Topically administered CBD is likely to also reach human HFs, which express functional TRPV4 receptors, the stimulation of which induces premature catagen (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), whereas adenosine reportedly promotes human hair growth (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 (23) Google Scholar). Therefore, we asked in the current exploratory pilot study whether and how CBD impacts on microdissected, organ-cultured human scalp 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 (58) Google Scholar) and primary human outer root sheath keratinocytes (ORSKs) isolated and cultured from plucked hair shafts (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 (5) Google Scholar), obtained after written informed consent from dermatologically healthy individuals. The study adhered to Helsinki guidelines, and was institutionally and governmentally approved. For methodological details, see Supplementary Materials and Methods. First, we probed if CBD influenced hair shaft production of microdissected human HFs. We found that, when applied at 0.1 μM, CBD tended to promote hair shaft elongation, but, unexpectedly, did not modulate HF cycling and hair matrix keratinocyte proliferation or apoptosis ex vivo (Figure 1). This tendency may represent a pseudo-elongation of the hair shaft, which is pushed out of its anchorage in the HF by tissue shrinkage during the course of organ culture. However, 10 μM CBD significantly suppressed hair shaft production (Figure 1a and Supplementary Figure S1), and induced development of catagen morphology (Figure 1b and c). This was accompanied by a significant reduction of the ratio of proliferating (Ki-67+), and a significant increase in the percentage of apoptotic (TUNEL+) hair matrix keratinocytes (Figure 1d and e), indicating the onset of an apoptosis-associated involution. This invited the hypothesis that CBD may concentration-dependently target different receptors. This hypothesis was probed in cultured ORSKs, which facilitate mechanistic analyses and permit higher throughput assessments than very scarcely available scalp HFs. In line with the above HF data, CBD rapidly decreased the ORSK count (3- to 72-hour treatments; CyQUANT assay; Supplementary Figure S2a-d), reduced viability, and induced chiefly apoptotic cell death in a concentration- and time-dependent manner (24-hour treatments; MTT assay and combined 1,1′,3,3,3′,3′-hexamethylindodicarbo-cyanine iodide (DilC1(5))-SYTOX Green labeling; Supplementary Figure S3a and b). CBD activates the mostly calcium-permeable TRPV4 ion channels (Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (76) Google Scholar), the stimulation of which promotes catagen in human HFs ex vivo (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). In line with this, the catagen-promoting CBD concentration (≥10 μM) induced calcium influx in cultured ORSKs (Figure 2a and b), which was almost completely abrogated by the highly selective TRPV4 antagonist, HC067047 (1 μM) (Everaerts et al., 2010Everaerts W. Zhen X. Ghosh D. Vriens J. Gevaert T. Gilbert J.P. et al.Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis.Proc Natl Acad Sci USA. 2010; 107: 19084-19089Crossref PubMed Scopus (264) Google Scholar; Figure 2c and d). This suggests that, at micromolar concentrations, CBD activates TRPV4 in human ORSKs, and that its catagen-promoting effect on organ-cultured HFs may be stimulated via signaling through this newly identified catagen-inducing pathway. Reportedly, toll-like receptor 3 activation on human ORSKs leads to inflammasome activation and subsequent IL-1β production (Shin et al., 2017Shin J.M. Choi D.K. Sohn K.C. Kim S.Y. Min Ha J. Ho Lee Y. et al.Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles.Sci Rep. 2017; 7: 44127Crossref PubMed Scopus (11) Google Scholar), which may create a proinflammatory, catagen- and HF dystrophy-promoting signaling milieu (Hoffmann et al., 1998Hoffmann R. Happle R. Paus R. Elements of the interleukin-1 signaling system show hair cycle-dependent gene expression in murine skin.Eur J Dermatol. 1998; 8: 475-477PubMed Google Scholar, Philpott et al., 1996Philpott M.P. Sanders D.A. Bowen J. Kealey T. Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata.Br J Dermatol. 1996; 135: 942-948Crossref PubMed Scopus (141) Google Scholar, Rückert et al., 2000Rückert R. Lindner G. Bulfone-Paus S. Paus R. High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo.Br J Dermatol. 2000; 143: 1036-1039Crossref PubMed Scopus (65) Google Scholar). Since CBD exerts remarkable anti-inflammatory effects in several systems (Esposito et al., 2013Esposito G. Filippis D.D. Cirillo C. Iuvone T. Capoccia E. Scuderi C. et al.Cannabidiol in inflammatory bowel diseases: a brief overview.Phytother Res. 2013; 27: 633-636Crossref PubMed Scopus (47) Google Scholar, Oláh et al., 2017Oláh A. Szekanecz Z. Bíró T. Targeting Cannabinoid Signaling in the Immune System: "High"-ly Exciting Questions, Possibilities, and Challenges.Front Immunol. 2017; 8: 1487Crossref PubMed Scopus (58) Google Scholar), we also assessed how a noncytotoxic (Supplementary Figures S2a-d and S3a and b) and noncatagen-promoting (Figure 1a-e) CBD concentration (i.e., 0.1 μM) impacts on the production of catagen-promoting cytokines by ORSKs. Interestingly, CBD effectively antagonized not only the toll-like receptor 3-activator polyinosinic-polycytidylic acid-induced upregulation of IL-1β, but also that of IL-6, IL-8, and tumor necrosis factor-α (Figure 2e). Similar to sebocytes, these anti-inflammatory actions of CBD appear to primarily depend on adenosine receptor-mediated signaling, because the adenosine receptor pan-antagonist CGS 15943 (Ghai et al., 1987Ghai G. Francis J.E. Williams M. Dotson R.A. Hopkins M.F. Cote D.T. et al.Pharmacological characterization of CGS 15943A: a novel nonxanthine adenosine antagonist.J Pharmacol Exp Ther. 1987; 242: 784-790PubMed Google Scholar, Klotz, 2000Klotz K.N. Adenosine receptors and their ligands.Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 382-391Crossref PubMed Scopus (274) Google Scholar, Williams et al., 1987Williams M. Francis J. Ghai G. Braunwalder A. Psychoyos S. Stone G.A. et al.Biochemical characterization of the triazoloquinazoline, CGS 15943, a novel, non-xanthine adenosine antagonist.J Pharmacol Exp Ther. 1987; 241: 415-420PubMed Google Scholar) almost completely abrogated the CBD effects reported above (Figure 2e). Besides potentially reducing the hair growth-inhibitory effects of the likely high level of these wounding-associated cytokines of freshly microdissected HFs compared with vehicle-treated controls, the moderate hair shaft-stimulatory effects of 0.1 μM CBD (Figure 1a) may also result from increased fibroblast growth factor-7 transcription by human dermal papilla fibroblasts (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). Therefore, whether submicromolar CBD helps to normalize a catagen-promoting proinflammatory HF signaling milieu, reduces the cytokine-driven recruitment and activation of hair growth inhibitory immune cells, and promotes the production of hair growth-supporting growth factors such as fibroblast growth factor-7 deserves further investigation. Although our pilot data need to be followed up by systematic dose-response experiments in a wider range of CBD concentrations, tested in HF organ cultures from additional patients, the current data invite the hypothesis that submicromolar, adenosine receptor-activating doses of CBD may reduce the intrafollicular production of catagen-promoting cytokines and could thus become useful as adjunct therapy for inflammatory hair loss disorders characterized by excessive levels of these cytokines. Instead, TRPV4-activating micromolar concentrations of CBD are attractive candidate inhibitors of unwanted hair growth (Supplementary Figure S4). If the current ex vivo findings, which imitate a systemic mode of CBD application, are reproducible after topical application, and if administering submicromolar versus micromolar topical CBD can indeed evoke differential, therapeutically desired effects in clinical hair growth management, these results deserve rigorous exploration. Detailed discussion about the limitations of this study can be found in Supplementary Discussion. The data that support the findings of this study are available from the corresponding authors upon reasonable request. Imre Lőrinc Szabó: https://orcid.org/0000-0002-9628-4372 Erika Herczeg-Lisztes: https://orcid.org/0000-0002-8517-6536 Gabriella Béke: https://orcid.org/0000-0003-3113-2287 Kinga Fanni Tóth: https://orcid.org/0000-0002-5184-8082 Ralf Paus: https://orcid.org/0000-0002-3492-9358 Attila Oláh: https://orcid.org/0000-0003-4122-5639 Tamás Bíró: https://orcid.org/0000-0002-3770-6221 AO and TB provide consultancy services to Botanix Pharmaceuticals Ltd (AO) and Phytecs Inc (TB). Botanix Pharmaceuticals Ltd, Phytecs Inc, 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. This project was supported by Hungarian research grants ( NKFIH 120552 , 121360 , and 125055 ; GINOP-2.3.2-15-2016-00015 "I-KOM Teaming"), and 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 János Bolyai Research Scholarship of the Hungarian Academy of Sciences and by the New National Excellence Program of the Ministry of Human Capacities ( ÚNKP-18-4-DE-247 ). KFT's work was supported by the Hungarian Ministry of Human Capacities ( NTP-NFTÖ-18-B-0168 ). The authors are grateful to Dániel Bereczki and to Erika Hollósi for their technical support. Conceptualization: RP, AO, TB; Formal Analysis: ILS, EL; Funding Acquisition: AO, TB; Investigation: ILS, EL, GB, KFT; Project Administration: ILS, EL, GB, KFT; Supervision: AO, TB; Validation: ILS, EL; Visualization: ILS, EL; Writing - Original Draft Preparation: ILS, AO; Writing - Review and Editing: ILS, EL, GB, KFT, RP, AO, TB. (–)-Cannabidiol (CBD), the TRPV4-selective antagonist HC067047 (Armstrong et al., 2015Armstrong J.L. Hill D.S. McKee C.S. Hernandez-Tiedra S. Lorente M. Lopez-Valero I. et al.Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.J Invest Dermatol. 2015; 135: 1629-1637Abstract Full Text Full Text PDF PubMed Google Scholar; http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?tab=biology&ligandId=4213), and the adenosine receptor pan-antagonist CGS 15943 (Ghai et al., 1987Ghai G. Francis J.E. Williams M. Dotson R.A. Hopkins M.F. Cote D.T. et al.Pharmacological characterization of CGS 15943A: a novel nonxanthine adenosine antagonist.J Pharmacol Exp Ther. 1987; 242: 784-790PubMed Google Scholar, Klotz, 2000Klotz K.N. Adenosine receptors and their ligands.Naunyn Schmiedebergs Arch Pharmacol. 2000; 362: 382-391Crossref PubMed Scopus (274) Google Scholar http://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=384) were purchased from Tocris (Bristol, United Kingdom), whereas the toll-like receptor 3 activator double-stranded RNA polyinosinic-polycytidylic acid was obtained from Sigma-Aldrich (St Louis, MO). CBD was dissolved in absolute ethanol, whereas the solvent of HC067047 and CGS 15943 was DMSO. Polyinosinic-polycytidylic acid was dissolved in filtered distilled water. Control cultures were always treated with appropriate amount of vehicle(s). Human skin samples were obtained following obtaining written informed consent from healthy individuals undergoing dermatosurgery. The study adhered to Helsinki guidelines, and was institutionally and governmentally approved by 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, and IF-778-5/2017). Human anagen VI hair follicles (HFs) were isolated from skin obtained from two male (28 and 32 years old) and one female (58 years old) patients undergoing neurosurgical intervention as described previously (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 (70) 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 (58) 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 (12) Google Scholar). Isolated HFs were cultured in William's E Medium (Invitrogen, Paisley, United Kingdom) supplemented with 2 mM L-glutamine (Invitrogen), 10 ng/ml hydrocortisone, 10 mg/ml insulin, and antibiotics (all from Sigma-Aldrich). Treatments were initiated 24 hours after the isolation. Medium was changed every other day, whereas treatment with CBD (or vehicle) was performed daily. Length measurements were performed on individual HFs using a light microscope with an eyepiece measuring graticule. Isolation and culture of outer root sheath keratinocytes (ORSKs) was performed following our previously optimized protocols (see, e.g., 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 (70) 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 (5) Google Scholar). In brief, plucked hair shafts of healthy volunteers (both females and males; aged 23–47 years old) were digested using trypsin to isolate ORSKs. Isolated ORSKs were seeded to a feeder layer of primary human dermal fibroblasts that were pretreated overnight with mitomycin C (0.4 μg/ml from Sigma-Aldrich) to block their proliferation. Human dermal fibroblasts were obtained from de-epidermized dermis using enzymatic digestion. Cocultures of nonproliferating feeder human dermal fibroblasts and ORSKs were maintained in a 1:3 mixture of Ham's F-12 and DMEM (both from Invitrogen) supplemented with 0.1 nM cholera toxin, 5 mg/ml insulin, 0.4 mg/ml hydrocortisone, 2.43 mg/ml adenine, 2 nM triiodothyronine, 10 ng/ml EGF, 1 mM ascorbyl-2-phosphate, and antibiotics (all from Sigma-Aldrich). ORSKs were plated for subsequent experiments on collagen-coated (1%; 37 °C for 1 hour; obtained from Sigma-Aldrich) culture dishes without human dermal fibroblast feeder layer. Cryosections of 6 μm thickness of the HFs were dried at room temperature (10 minutes), and fixed in precooled acetone (10 minutes at −20 °C). Sections were then washed in distilled water for 1 minute at room temperature, and stained with Mayer's hematoxylin for 35 seconds at room temperature. Following gentle washing under the tap water for 15 minutes at room temperature, sections were stained with 0.1% Eosin Y solution (1 minute at room temperature). Sections were then dehydrated (washing three to four times in 70%, 96%, and 100% ethanol), and incubated in xylene (2 × 10 minutes at room temperature). Finally, sections were mounted by using Eukitt quick-hardening mounting medium (Sigma-Aldrich). Images were taken by using bright-field function of a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan). Hematoxylin and eosin staining was then used for studying HF morphology, and, together with Ki-67/TUNEL double labeling , for staging hair cycle to anagen, early catagen, and late catagen according to predefined objective morphological criteria (Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N.V. 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 (94) Google Scholar). Proliferation and apoptosis were assessed by Ki-67/TUNEL double immunostaining as described previously (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 (70) Google Scholar, Kloepper et al., 2010Kloepper J.E. Sugawara K. Al-Nuaimi Y. Gáspár E. van Beek N.V. 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 (94) 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 (12) Google Scholar). In brief, cryosections (6 μm) were air dried for 10 minutes at room temperature, and then fixed for 10 minutes in 1% paraformaldehyde in phosphate-buffered saline (PBS) (15 mM NaCl, 20 mM Na2HPO4 [pH 7.4]; all from Sigma-Aldrich) at room temperature. Samples were rehydrated in PBS for 5 minutes (2×), and were then postfixed for 5 minutes in precooled ethanol–acetic acid 2:1 at −20 °C, and then rehydrated in PBS again for 5 minutes (3×). After rehydration, sections were incubated with an equilibration buffer for 5 minutes at room temperature, and then incubated with TdT enzyme for 1 hour at 37 °C. To reveal proliferation, mouse anti-human Ki-67 (DAKO, Carpinteria, CA) primary antibody was used. In addition, to assess apoptosis, a TUNEL assay was performed using an ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore) following the manufacturer's instructions. Sections were incubated with the primary antibody overnight at 4 °C, and then with the secondary antibody (Alexa Fluor 568 Goat anti-Mouse IgG; Invitrogen) for 45 minutes at room temperature. Sections were washed in PBS and were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromont-G (Southern Biotechnology Associates, Birmingham, AL). Visualization of the signals was performed using a Nikon Eclipse E600 fluorescent microscope (Nikon). The ratio of proliferating (Ki-67+) and apoptotic (TUNEL+) cells was determined in defined reference regions of the hair bulb, and are expressed as the percentage of the total cell count determined by counting DAPI+ nuclei. Quantitative real-time PCR was performed on a Stratagene MXP3005p detection system (Agilent Technologies, Santa Clara, CA) using the 5ʹ nuclease assay, as we have described previously (Oláh et al., 2014Oláh A. Tóth B.I. Borbíró I. Sugawara K. Szöllõsi A.G. Czifra G. et al.Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes.J Clin Invest. 2014; 124: 3713-3724Crossref PubMed Scopus (76) Google Scholar). In brief, ORSKs were seeded to Petri dishes (d = 35 mm; 500,000 cells/1.5 ml medium/dish), and were harvested following the indicated 3-hour treatments. Total RNA was then isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The concentrations and purities of the RNA samples were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Budapest, Hungary), and samples were kept at −20 °C until further processing. Next, DNase treatment was performed according to the manufacturer's protocol, and, then, 3 μg of total RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Kit from Life Technologies Hungary, Budapest, Hungary. PCR amplification was performed using the TaqMan Gene Expression Assays (assay IDs: Hs00174097_m1 for IL-1α; Hs00985639_m1 for IL-6; Hs00174103_m1 for IL-8; and Hs00174128_m1 for tumor necrosis factor-α) and the TaqMan Universal PCR Master Mix protocol (Applied Biosystem, Waltham, MA). As internal control, transcripts of glyceraldehyde 3-phosphate dehydrogenase (assay ID: Hs99999905_m1) were determined. The amount of the transcripts was normalized to those of the housekeeping gene using the ΔCT method, and then the relative expression values were further normalized those of the vehicle-treated control (ΔΔCT method). ORSKs were seeded in 96-well black well plates with clear bottom (Greiner Bio-One, Kremsmuenster, Austria) previously coated with 1% collagen in ORSK medium at a density of 20,000 cells per well, and incubated at 37 °C for 24 hours. The cells were washed once with 10 mg/ml BSA and 2.5 mM probenecid (both from Sigma-Aldrich) containing Hank's solution (136.8 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 0.81 mM MgSO4, 1.26 mM CaCl2, 5.56 mM glucose, 4.17 mM NaHCO3 [pH 7.2], all from Sigma-Aldrich), and then were loaded with 1 μM Fluo-4 AM (Life Technologies Hungary ) dissolved in Hank's solution (100 μl/well) at 37 °C for 30 minutes. The cells were then washed three times with Hank's solution (100 μl/well). The plates were then placed into a FlexStation II384 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA) and alterations of the cytoplasmic calcium concentration (reflected by changes in fluorescence; λEX = 490 nm, λEM = 520 nm) were monitored at room temperature following the application of the indicated substances or vehicle. To probe reactivity and viability of the cells, at the end of each measurement, 0.2 mg/ml adenosine triphosphate was administered as a positive control (data not shown). Data are presented as F/F0, where F0 is the average baseline fluorescence (i.e., before compound application), whereas F is the actual fluorescence. The degree of cellular growth (reflecting proliferation) was determined by measuring the DNA content of cells using a CyQUANT Cell Proliferation Assay Kit (Life Technologies Hungary). ORSKs (5,000 cells per well) were cultured in 96-well black well plates with clear bottom (Greiner Bio-One), and were treated as indicated in octuplicates. Supernatants were then removed by blotting on paper towels, and the plates were subsequently frozen at −80 °C. The plates were then thawed at room temperature, and 200 μl of CyQUANT dye/cell lysis buffer mixture was added to each well. After 5 minutes of incubation, fluorescence was measured at 490 nm excitation and 520 nm emission wavelengths using a FlexStation II384 Multi-Mode Microplate Reader (Molecular Devices). Relative fluorescence values were expressed as the percentage of the daily vehicle control regarded as 100%. To assess the effect on viability, we applied an MTT assay (Sigma-Aldrich) because mitochondrial enzymes of living cells are able to transform tetrazolium salt (MTT) into formazan crystals. ORSKs were seeded in 96-well plates (density: 10,000 cells/well) and were treated as indicated in octuplicates with various concentrations of CBD or vehicle. Cells were then incubated with 0.5 mg/ml MTT reagent for 3 hours, and the concentration of formazan crystals (as an indicator of number of viable cells) was determined colorimetrically at 565 nm by using a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices). Results are expressed as percentage of vehicle control regarded as 100%. A decrease in the mitochondrial membrane potential is one of the earliest markers of apoptosis (Green and Reed, 1998Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar; Susin et al., 1998). Therefore, to assess the process, the mitochondrial membrane potential of ORSKs was determined using a MitoProbe DilC1(5) Assay Kit (Life Technologies Hungary). Cells (10,000 cells/well) were cultured in 96-well black well plates with clear bottom (Greiner Bio-One) in octuplicates and were treated as indicated. After removal of supernatants, cells were incubated for 30 minutes with DilC1(5) working solution (50 μl/well), then washed with PBS, and the fluorescence of DilC1(5) was measured at 630 nm excitation and 670 nm emission wavelengths using a FlexStation II384 Multi-Mode Microplate Reader (Molecular Devices). Relative fluorescence values were expressed as the percentage of vehicle control regarded as 100%. Necrotic processes were determined by SYTOX Green staining (Life Technologies Hungary). The dye is able to penetrate (and then to bind to the nucleic acids) only necrotic cells with ruptured plasma membranes, whereas healthy cells with intact surface membranes show negligible SYTOX Green staining intensity. Cells were cultured in 96-well black well plates with clear bottom (Greiner Bio-One), and treated as indicated. Supernatants were then discarded, and the cells were incubated for 30 minutes with 1 μM SYTOX Green dye. Following incubation, cells were washed with PBS, the culture medium was replaced, and fluorescence of SYTOX Green was measured at 490 nm excitation and 520 nm emission wavelengths using a FlexStation II384 Multi-Mode Microplate Reader (Molecular Devices). Relative fluorescence values were expressed as the percentage of negative control regarded as 100%. Owing to their spectral properties, DilC1(5) and SYTOX Green dyes were always administered together, enabling us to investigate necrotic and early apoptotic processes of the same cultures. Selective decrease of DilC1(5) intensity indicated mitochondrial depolarization (i.e., the onset of early apoptotic processes), whereas increase of SYTOX Green staining intensity revealed necrotic cell death. When applicable, data were analyzed using OriginPro Plus 6 software (Microcal, Northampton, MA) using chi-square test followed by Fisher's exact test, Student two-tailed unpaired t-test, or analysis of variance with Bonferroni post hoc test, and P < 0.05 values were regarded as significant differences. When interpreting the results presented in the current exploratory, pilot study, one has to keep in mind that, although it sheds light on previously unknown and potentially relevant (hair-wise) dermatological effects of CBD, the current work has certain important limitations as well. First and foremost, as mentioned in the main text, our data need to be followed up by systematic dose-response experiments in a wider range of CBD concentrations, tested in HF organ cultures from additional patients to explore the existence and magnitude of inter-donor variability. Second, in the current study, by adding CBD (or vehicle) to the culture medium we mimicked the effects of systematically administered CBD. However, from a translational point-of-view it is crucially important to assess the biological effects of CBD following standardized topical application in appropriate formulations. Third, it is important to emphasize that, in the current study, we used microdissected, truncated HFs lacking the bulge region as well as other (non-HF) members of the pilosebaceous unit (namely the sebaceous glands and the arrector pili muscles). Therefore, we cannot exclude the possibility that specific (and most likely concentration dependent) actions of CBD on these structures may influence the production and release of hair cycle-regulating substances, further modifying its already quite complex effects on the hair cycle. Fourth, studying microdissected HFs ex vivo has the inherent limitation of missing the effects and regulatory roles of intact innervation and blood supply. This may be crucially important, because it can lead to model dependently opposing observations in certain cases. In the case of, for example, the TRPV1-activating capsaicin, we have previously shown that it suppressed hair growth and induced premature catagen entry in microdissected human HFs (Bíró et al., 2006Bíró T. Bodó E. Telek A. Géczy T. Tychsen B. Kovács L. et al.Hair cycle control by vanilloid receptor-1 (TRPV1): evidence from TRPV1 knockout mice.J Invest Dermatol. 2006; 126: 1909-1912Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Moreover, lack of TRPV1 in knockout animals delayed their normal hair cycle (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 Scopus (160) Google Scholar). However, administration of capsaicin was found to promote hair growth by increasing IGF-I production in mice and in humans with alopecia, most likely by acting on sensory nerve fibers (Harada et al., 2007Harada N. Okajima K. Arai M. Kurihara H. Nakagata N. Administration of capsaicin and isoflavone promotes hair growth by increasing insulin-like growth factor-I production in mice and in humans with alopecia.Growth Horm IGF Res. 2007; 17: 408-415Crossref PubMed Scopus (49) Google Scholar). Given the broad target spectrum of CBD, one can speculate that it may also modulate production and release of hair cycle-regulating substances from sensory nerve endings. Therefore, its impact on human hair biology may be even more complex.

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