by Kevin Rands | October 25, 2004 9:05 am
Long-Term (5 Years) Multinational Experience with Finasteride 1-mg in the Treatment of Men with Androgenetic Alopecia (Male Pattern Hair Loss)
Keith D. Kaufman, for The Finasteride Male Pattern Hair Loss Study Group, Merck Research
Laboratories, Rahway, NJ, USA
We previously demonstrated the safety and efficacy of finasteride in the treatment of men with male pattern hair loss based on pooled 2-year data from two large, replicate, double-blind, placebo-controlled Phase III studies. The present analysis assessed the pooled long-term, placebo-controlled safety and efficacy data through 5 years in these studies. In two 1-year Phase III trials, 1553 men aged 18 to 41 years with male pattern hair loss were randomized to receive oral finasteride 1 mg/day or placebo for 1 year, and 1215 men continued in up to 4 consecutive, 1-year, placebo-controlled, blinded extension studies. Efficacy was evaluated by scalp hair counts, patient and investigator assessments, and global photographic assessment by an expert panel. Finasteride treatment led to improvements in scalp hair compared to baseline and placebo by all evaluation techniques during 5 years of treatment (p < 0.001 vs. placebo). Improvements in hair count (baseline = 876 hairs), measured in a 1-inch diameter circular area (5.1 cm2) of balding vertex scalp, were observed with finasteride compared with placebo (difference of 126, 171, 183, 219 and 277 hairs at 1, 2, 3, 4 and 5 years, respectively; p < 0.001 for all between-group comparisons). Treatment with placebo led to progressive hair loss over 5 years. Patient self-assessment demonstrated that finasteride treatment led to improvements in hair growth, appearance of hair, and satisfaction with appearance of hair. These improvements were corroborated by the investigators’ assessments of patients. Global photographic assessments demonstrated the superiority of finasteride over placebo and confirmed that, without treatment, progressive visible hair loss occurs. Finasteride was generally well tolerated in the studies. Side effects were minimal and no new side effects were observed with increasing duration of exposure to finasteride. In men with male pattern hair loss, treatment with finasteride 1 mg/day was generally well tolerated and led to a durable reduction in the progression of hair loss and improvements in hair growth over 5 years.
Irreversibility of hair follicle changes after 30 months of Androgenetic Alopecia.
Konstantinova N, Korotkii N.G, Sharova N, Barhunova E, Gaevski D.
Nioxin Research Inc, Atlanta, USA
Moscow Medical University
We studied horizontal and vertical biopsy from 15 Caucasian 24-41 year old males diagnosed with bitemporal recession Androgenetic Alopecia (AA) for 1.5 –18 years (average 7.4 years). All 15 biopsies were stained with H&E, Van Gieson and with other collagen specific stainings. 1. Eleven pts with AA longer than 3 years had perifollicular fibrosis – collagen fibers were compact and formed a small scar-like formation around each anagen hair follicle(HF). Two patients – 33 year old with 18 month AA and 23 year old with 20 month AA did not have these hair follicle changes. Two 26-year-old patients with 30 and 36 month AA respectively were found to have some not so severe collagen fiber changes. 2. Infundibulum of HF dilatated 124-192 mm and most of them covered with keratinazed plug lacking normal hair shaft growth. 3. Decreased number of hair follicles 1.75-2,45 per sq. mm from 3.5-5 per sq. mm in control group. 4. None of anagen HF was situated in subcutaneous fat. We showed a correlation between length of the AA and severity/ thickness of perifollicular fibrosis. The result of this study is that any treatment of AA is recommended to start earlier than 30 months from first signs of AA. This should prevent irreversible collagen changes associated with “fibrotic incapsulation” of most anagen HF in involved areas, which usually leads to loss of normal blood supply, innervation, and subsequent miniaturization and prevention of hair from normal cycling.
Effects of Finasteride on Apoptosis and Regulation of the Human Hair Cycle.
Department of Physiology and Biophysics, Miami, FL, USA.
A number of studies have provided evidence that apoptosis is a central element in the regulation of hair follicle regression. In androgenetic alopecia (AGA) the exact location and control of key players in the apoptotic pathways remains obscure. In the present study, we used a panel of antibodies and investigated the spatial and cellular pattern of expression of caspases and Inhibitors of Apoptosis (IAPs) such as XIAP and FLIP, in men with normal scalp and in men with AGA before and after 6 months treatment with 1 mg oral finasteride treatment. Constitutive expression of caspases-1, -3, -8 and -9 and XIAP was detected predominately within the isthmic and infundibular hair follicle area, basilar layer of the epidermis, and eccrine and sebaceous glands. AGA affected tissues showed an increase in caspase (-1, -3, -6, -9) immunoreactivity with a concomitant decrease in XIAP staining. After 6 months of finasteride treatment both caspases and XIAP were similar to levels exhibited by normal subjects. Immunoblot analysis was performed to determine antibody specificity and cellular expression of caspases. Purified populations of keratinocytes, melanocytes, dermal papilla, dermal fibroblasts derived from human hair follicles were cultured in vitro and treated with 0.5 mm staurosporin. Time-course experiments revealed that processing of caspase-3 is a principal event during apoptosis of these hair cell types. These data suggest that alterations in levels of caspases and IAPs regulate hair follicle homeostasis. Moreover, finasteride appears to influence caspase and XIAP expression in hair follicle cells thus signaling anagen, active growth in the hair cycle.
Treatment of androgenetic alopecia with combination therapy of oral finasteride, topical minoxidil, and tretinoin.
Gwang Seong Choi, Sung Joo Lee.
Department of Dermatology, Inha University, Inchon, Korea
Finasteride is an effective oral medication in the treatment of androgenetic alopecia (AGA). Minoxidil has a direct effect on hair growth by stimulating dermal papillae or follicular hair matrix cells. Simultaneous administration of topical minoxidil with tretinoin may enhance the response of AGA to minoxidil. We treated 41 AGA patients from 23 to 43 years of age (grade III and VI, according to the Norwood-Hamilton classification scale) with combination therapy. The results were evaluated by patient self assessment and investigator assessment by comparision of clinical photography. Every 3 months, clinical evaluation and adverse events were assessed. At month 12, of the 41 patient enrolled, Effective results, including excellent results and good results, were observed in 27 (65.9%) patients. Progressed hair loss compared to baseline were 2 (4.9%) patients. Adverse effects including sexual dysfunctions and skin irritation were observed in 2 (4.9%) and 6 (14.6%) patients, respectively. The results between self assessment and investigator assessment showed similarity. The combination therapy of oral finasteride 1mg/day, topical 3% minoxidil, and 0.025% tretinoin improved AGA more efficiently comparing to monotherapy. We recommended combination therapy in those patients with AGA who desire medical therapy.
Androgenetic alopecia and its relationship with testicular cancer
MP Birch, JCRBowling, AG Messenger
Departments of Dermatology, Royal Hallamshire Hospital, Sheffield & The Middlesex Hospital, London, UK
During recruitment for a clinical trial of a treatment for androgenetic alopecia in young men, two out of 30 men wishing to enrol in the trial gave a history of testicular cancer. We therefore carried out an observational study to examine the association between male androgenetic alopecia and testicular cancer in more detail. We examined 144 patients with a diagnosis of testicular cancer attending an oncology clinic. They were classified by: histological diagnosis (68 teratoma, 73 seminoma, 2 mixed seminoma/teratoma and 1 leydig cell), age, treatment received, and hair status (Norwood-Hamilton Scale). Hair status was assessed by two observers and compared with a database of 558 healthy males drawn from the local population and in whom hair status had been evaluated by the same observers. Hair loss was classified as nil (Norwood Hamilton grades I and II), mild (grades IIv, III, IIIa, IIIv), moderate (grades IV and V) and severe (grades VI and VII). There was a significant increase in mild hair loss in men with seminoma (52%) compared with controls (25%) whether adjusted (p<0.01, confidence interval 1.97-7.44; Mantel-Haenszel test) or unadjusted (p<0.01, CI 1.57-5.74) for age. There was a significant increase in mild hair loss in men with teratoma (38%) compared with controls (25%) when adjusted (p<0.01, CI 1.23-4.88) for age, however this was not significant when unadjusted for age (p=0.25, CI 0.76 – 2.52). The causes of testicular cancer are unknown but genetic factors, cryptorchidism, exposure to maternal hormones in utero and exposure to other environmental hormones have all been implicated. Our results indicate that balding tends to occur earlier in men predisposed to seminoma suggesting that similar genetic or environmental factors underlie both conditions.
Perception of Men with Androgenetic Alopecia by Women and Nonbalding Men
Dept. of Dermatology and Biostatistics
College of Medicine, The Catholic University of Korea, Seoul, Korea
Male pattern hair loss has significant negative psychosocial effects. It has been asserted that the negative effect of AGA is often trivialized by those not affected by it, and most studies concerning the perception of balding have focused on the effects on the patients. In this study, we assessed the perception of balding men by others. A questionnaire on the perception of balding men was answered in 90 nonbalding and 30 balding men and 130 women. Balding men were perceived as being older and less attractive in over 90% of the total subjects. However, a perception of being less confident, duller, and less potent was reported by less than half of the subjects. A perception of looking less attractive was significantly more common in women than nonbalding men (p<0.05). A perception of less confidence was significantly more common in balding men than nonbalding men (p<0.05). The perception of men with AGA by others was similar to the psychosocial effects reported by the patients themselves. This suggests that AGA can significantly influence social interactions.
Investigation of the Systemic Bioavailability of 5% Minoxidil Topical Solution in Young Males with Early Androgenetic Alopecia
Pharmacia Consumer Healthcare, Peapack, NJ, USA and TKL Research Inc., Paramus, NJ
There are no pharmacokinetic data on minoxidil topical solution (MTS) in adolescents with androgenetic alopecia (AGA). The objective of this investigation was to establish the steady-state percutaneous absorption and systemic bioavailability characteristics of 5% MTS in young male subjects (age 17 years and younger) with early AGA. This study was designed as a single-arm, single-center, open-label clinical investigation. Thirteen male subjects, age 17 or younger, with evidence of thinning hair at the frontal or vertex area of the scalp, self-administered 5% MTS 1.0 mL twice daily for a total of 11 doses to the balding area of the scalp. Serum and urine minoxidil levels were measured. AUC estimation and Cmax calculations were performed. Thirteen subjects who met entry criteria completed the study and were evaluable for analysis. Steady-state levels of minoxidil were achieved rapidly in these subjects. Mean Cmax was 1.58 ng/mL and mean AUC (0-24) was 25.4 ng-hr/mL. Mean total minoxidil excretion over the initial 12 hours after the application of the last dose was 537 mcg. There were no reports of scalp irritation with the use of 5% MTS. Fourteen adverse events were reported including abnormal liver function test findings. One subject had 2 adverse events (increased total bilirubin and increased ALT) that the investigator attributed to the study medication; none were serious. No one discontinued study participation because of an adverse event. Based on the results of this study, and in comparison to previous studies in older male subjects, it appears that younger subjects (
Incidence of Androgenetic Alopecia in Males 15 to 17 Years of Age
Pharmacia Consumer Healthcare, Peapack, NJ, USA and Q2 Marketing Research, Inc., Cincinnati
The incidence of androgenetic alopecia (AGA) among males 15 to 17 years of age has not been well established. The objective of this study was to determine the incidence of AGA among a large sample of 15- to 17-year-old males. Males who were 15 to 17 years of age (inclusive) were independently screened, recruited, and then interviewed by 10 board certified dermatologists at their respective research facility. Each participant filled out a self-administered questionnaire and underwent a scalp examination by the dermatologist. A modified version of the Hamilton-Norwood Scale was used to evaluate each subject. Five hundred and seventy-six adolescents agreed to participate; 496 (86%) actually responded and were enrolled. Dermatologists found that among those subjects who participated in the survey, 16% exhibited signs of AGA. Additionally, dermatologists judged that 14% of the respondents exhibited early signs of AGA. About one third of the respondents have a history of thinning hair or baldness on their mother’s side of the family while over one half have a history of thinning hair or baldness on their father’s side of the family. Very few respondents examined had evidence of other underlying scalp conditions or disorders. This study provides a reasonable estimate of the incidence of AGA in males 15 to 17 years of age. The true incidence of AGA among males in this age range is estimated to be within the range of the two rates determined in this study: 16% (modified Hamilton-Norwood Scale) to 14% (dermatologist-reported early signs of AGA).
Stabilization of Hair Loss with Use of Minoxidil Topical Solution
Pharmacia Consumer Healthcare, Helsingborg, Sweden and Peapack, NJ, USA
A post-marketing surveillance study in over 11,000 minoxidil topical solution (MTS) 2% users with androgenetic alopecia demonstrated that 4 out of 5 subjects experienced a slowing or stopping of hair loss with MTS (Rogaine/Regaine). Although subjects’ opinion of treatment results were considered to be important, further corroboration of these results was conducted and other data have been generated to support stabilization of hair loss. A recent analysis of hair count data in four multicenter studies evaluating both MTS 2% and 5% (two studies in females and two in males) has substantiated that hair loss can be stabilized with MTS use. The hair count stabilization data, based on a calculation of increased or unchanged number of non-vellus hairs compared to baseline, showed that for males the stabilization (percent of subjects) was 90% and 77% for MTS 2% users, and 96% and 75% for MTS 5% users. For females, the percentages were 87% and 88% for MTS 2% users, and 85% and 89% for MTS 5% users. In addition, using hair count data in subjects treated with MTS 2% and 3% for up to 5 years (Olsen et al [JAAD 1990;22:643-46), it was estimated that hair loss stabilized in 4 out of 5 (80%) subjects. An evaluation of global photography, carried out by two blinded dermatology experts for a registration trial of MTS 5% in men, further supports stabilization of hair loss. One expert evaluated hair loss stabilization to be 94% in both MTS 2% and 5% MTS groups. The second expert reported stabilization in 85% of the subjects using MTS 2%, and 83% using MTS 5%. In conclusion, multiple studies support the ability of MTS to stabilize hair loss in 4 out of 5 users.
Clinician Survey Evaluating Minoxidil Topical Solution in the Treatment of Androgenetic Alopecia in Patients Under 18 Years of Age
Pharmacia Consumer Healthcare, Peapack, NJ, USA and TKL Research Inc., Paramus, NJ
The use of minoxidil topical solution (MTS) for the treatment of androgenetic alopecia (AGA) in patients less than 18 years of age has not been previously characterized. The objective of this study was to estimate the prevalence of use of MTS among patients under 18 years of age for the treatment of AGA. This survey of clinicians was conducted with a written questionnaire and supported by face-to-face interviews with respondents who claimed to have prescribed MTS for the treatment of AGA in patients less than 18 years of age. Data were collected from 84 clinicians who had treated a total of 448 patients between the ages of 10 and 18 years. The majority of the patients were male (76%); 24% were female. The mean ages of the patients at initiation of MTS treatment for their hair loss were 15.8 and 15.2 years, in males and females, respectively. In the males, 38% had frontal hair loss, 21% had vertex hair loss, and 40% had both frontal and vertex involvement. The mean duration of MTS treatment was 17.4 and 19.2 months, in males and females, respectively. Sixty-seven percent of males and 63% of females are currently still undergoing treatment for AGA with MTS. For patients whom the response to treatment was known, 55% of males and 51% of females showed an improvement in scalp coverage or reduced hair loss; 40% of males and 44% of females showed no change. Overall, only 5% of the patients had worsening of their hair loss. Very few patients (6% overall) reported adverse reactions that were related to MTS treatment (mostly itching or mild irritation). This study shows that there is a population of patients (ie, less than 18 years of age) who are being treated off-label with MTS. Furthermore, the results suggest that MTS use in patients less than 18 years of age has been effective, safe, and well-tolerated.
Effect on Hair Growth of HEM-13/HDC Hair Tonic (Herbal Extract Mixtures) in Androgenetic Alopecia as Measured by Phototrichogram
BJ Shin, SW Ahn, WJ Tak, BI Ro.
Dept. of Dermatology, College of Medicine, Chung Ang University, Seoul, Korea.
We studied to investigate the clinical improvement of herbal extract mixtures in the patients with androgenetic alopecia. Total 76 patients (treatment group: 62, placebo group: 14, male: 63, female: 13) with androgenetic alopecia received topical application of HEM-13/HDC hair tonic or placebo for 6 months and then all of them received topical application of HEM-13/HDC hair tonic for 3 months. The efficacy was evaluated by hair counts, patient self-assessment, global photographic assessment and phototrichogram of the bald area in a circle of 1cm in diameter. Hair count and image analysis was performed before treatment, at 3, 6, 9 month after treatment respectively to compare the hair shaft diameter and hair density. At baseline, mean total hair counts in the tested area were 63.4 in the treatment group and 64.7 in the placebo group. At month 6, patients in the placebo had a decrease (mean ± SE) in hair counts of 1.3 ± 0.4 hairs. At month 9, patients in the treatment had an improvement (mean ± SE) in hair counts of 3.5 ± 0.7 hairs respectively (p<0.05), and had a significant increase in hair diameter (p<0.05). Over 85% of the subjects showed improvement of clinical symptoms and increase of hair growth. These data support that HEM-13/HDC hair tonic results in favorable effects on hair growth of patients with androgentic alopecia. In Mice, Hair growth was assessed by photographic method. In both the HEM-13/HDC and minoxidil groups, the hair grew into the normal state in 3 weeks but in the vehicle group, hair grew only to 1/3 the state of HEM-13/HDC group. In the animal study, it is as effective as or more effective than minoxidil. Any skin reactions were not observed in clinical and animal studies. We concluded that HEM-13/HDC has a hair growth stimulating effect.
The Hair-Growing Activity of Procyanidin Oligomers
Department of Dermatology, Toyama Medical and Pharmaceutical University, Sugitani, Toyama, JP.
Procyanidins are a family of condensed tannins we have identified in apples, which act as a hair-growing factor in the murine model both in vitro and in vivo. We have previously reported that the growth-promoting effect on murine hair epithelial cells attributable to procyanidin B-2, one species of procyanidin oligomer, reaches about 300% relative to controls; and have also shown that procyanidin B-2 possesses intensive anagen-inducing activity in the C3H in vivo mouse model. This presentation describes our investigations during a 12-month clinical trial of highly purified procyanidin oligomers isolated from unripe apples, chiefly comprising procyanidin B-2, procyanidin B-1, and procyanidin C-1. The clinical trial was performed in a total of 21 subjects showing male pattern baldness on the head. The test agent (about 1.8 ml per dose) was applied to the subjects’ affected scalp area twice a day, giving a daily dose of 16 mg of procyanidin oligomers. During the 12 months of twice-daily application of the agent, the hair-growing effects were evaluated according to the following parameters: the macrophotographically recorded change in the number of hairs in the designated scalp area, the changes in the diameter of hairs clipped from the designated scalp area, and the changes in the photographically recorded global view of the subjects’ heads. No side effects were observed in any subjects. After 12 months of use, 71% of the subjects showed an increased number of hairs in the designated scalp area relative to pre-trial measurements. The numbers of total hairs in the designated scalp area after the 12-month trial were significantly greater than the measured values at the start of the trial (paired t-test, p < 0.005). We also observed a clear trend towards increased number of non-vellus hairs (> 40 µm) in the designated scalp area after the 12-month trial compared to the values measured at the start of the test. A number of the subjects showed cosmetically satisfactory changes. Procyanidin therapy shows promise as a potential cure for male pattern baldness.
Early Detection of Decreased Hair Numbers and Hair Miniaturization in Androgenetic Alopecia in Man.
T. Leroy and D. Van Neste.
Skinterface sprl, Tournai, Belgium and Hair Technology ®, Brussels, Belgium
In male androgenetic alopecia (AGA), global changes of scalp hair observed on many years are the result of discrete structural and/or functional modifications at the level of individual hair follicles. The aim of this study was to evaluate correlation between hair density, % of thin hair and clinical staging. 5 controls (C) and 21 untreated male subjects with AGA (16-51 years) were examined and classified according to a modified Norwood-Hamilton scale (9 stage I-II ; 9 stage III and 3 stage V). Scalp photographs (primary enlargement x 3) were taken in all subjects on 3 anatomical sites (fixed coordinates on the left and right side of the top of the head and one occipital). Hair counts were obtained from two different photographic procedures i.e. before and after contrast enhancement (CE). Percentages of thin hair (< 40µm) were obtained after microscopic measures on clipped hair. We observed more scalp hairs after contrast enhancement (p<0,0001). Significant differences of extra hair (n/cm 2) were detected specifically between groups (p=0,0002) on top of head (C +29; AGA +60) as opposed to the occipital site (C +29; AGA +33). On the top of the head, we observed a decrease of hair density with both photographic methods (controls > I -II = III > V; p<0,05) and an increase of thin hair (I -II = III < V; p<0,0001) in correlation with the Hamilton severity score while no changes were recorded in the occipital site. The extra 60 hairs observed after CE in AGA (top of head) reflects probably a mixed population of thin and less colored hair already engaged in the “miniaturisation process” usually undetected without CE. The density changes occur at an early stage of AGA while the top of the head is not yet clinically affected (severity grades I – III). The more drastic decreases of density in the more severely affected patients (V) are consistent with hair thinning and the clinical observation of an expanding wave of follicular hypotrophy.
Androgenetic Alopecia in Adolescence
Wilma Bergfeld, Fabiane Mulinari–Brenner
Department of Dermatology, The Cleveland Clinic Foundation, Cleveland, Ohio – USA
Androgenetic alopecia (AGA) is an autosomal dominant condition with variable penetrance that affects about 50% of men and women. AGA is androgen mediated with puberty onset (early teens or twenties) in both sexes and frequently being fully expressed by the forties. Early identification of this condition leads to better treatment results. A review of patients under 18 years old with clinical diagnosis of AGA was performed. These patients were seen from 1997 to 2000 on the Department of Dermatology – The Cleveland Clinic Foundation. Clinical presentation, family history of AGA, laboratory tests and scalp biopsies were reviewed. Twenty-one patients (4 female and 17 male) were in the young cohort. Age range was 13-17 years (mean 15.4 years). Frontal, vertex or both areas were affected. Family history of AGA was present in 18 patients. Androgen excess signs such as acne (7 patients), hirsutism (4 patients) and seborrheic dermatitis (7 patients) were also associated with alopecia. Hormone levels suggested androgen excess only in 6 patients (2 female and 4 male) of 16 tested. Scalp biopsies were performed in 3 patients and confirmed the presence of hair follicle miniaturization. AGA may start early in the adolescence, especially when the patient has family history of AGA. It can be associated with other signs of androgen excess, however laboratory tests are not always helpful. AGA should be remembered as a cause of hair loss in adolescence.
QUANTIFYING PROGRESSION OR REVERSAL OF FOLLICULAR MINIATURIZATION IN ANDROGENETIC ALOPECIA BY IMAGE ANALYSIS
David A. Whiting and Douglas Canfield
Baylor Hair Research and Treatment Center
Histologic analysis of horizontal sections of scalp biopsies in men and women with androgenetic alopecia is an established method of measuring progression of hair loss or hair regrowth. Detailed follicular counts are time consuming, making this technique problematic for large drug trials. Digital imaging can provide reproducible information which can be more precise than that derived from visual microscopic examination. A method of image analysis was developed to quantify follicular counts and to size individual follicles. A Nikon D1 digital camera was attached to an Olympus BX 40 microscope. The apparatus was linked to a Dell Dimension XPS T500 desktop computer. An imaging program was used, and data was handled with automated computer analysis. A large number of biopsies from different trials were studied. These biopsies had already been evaluated by visual histologic analysis with the observer blinded to time and treatment. All biopsies were re-photographed with the digital camera using a 2x magnification objective. The entire 4 mm cross-section of a biopsy was photographed. The images were stored and examined later with similar observer blinding. All terminal and vellus hairs visible in the papillary dermis were marked by the investigator. A technician then mapped each cross-sectional hair shaft area and automated image analysis was performed. Total hairs in each section were counted. Hairs were classified into four different groups. The cross-sectional size varied from vellus-like hairs through intermediate hairs and small terminal hairs to large terminal hairs. This quantification could yield an average hair diameter for the entire section and also enumerate the hairs of different diameters to reflect different stages of the miniaturization process. The results were analyzed in both active treatment and placebo groups. They could also be compared to the previous results from the standard visual follicular counts in histological sections. Details of these results will be presented.
HISTOLOGY AND HORMONAL ACTIVITY IN SENESCENT THINNING IN MALES
Price V.H., Dept. of Dermatology, University of California, San Francisco; Sawaya M.E., ARATEC Clinics & University of Miami, Miami, Florida; Headington J.T., Dept. of Dermatology & Pathology, Ann Arbor, MI; Kibarian M.K., Dept. of Dermatology, George Washington University, Washington, D.C.
Senescent thinning of the scalp hair, or thinning that occurs after age 60, is poorly understood, and it is unclear whether this is a distinct entity or part of the continuum of androgenetic alopecia (AGA). In a previous study, young males age 18 to 30 with AGA had higher levels of 5a-reductase type 1 and 2, more androgen receptors, and lower levels of cytochrome P-450 aromatase in hair follicles in the frontal region of the scalp than in the occipital region. This study in males age 60 years and older was designed to determine whether the histology and hormonal findings in older males with hair thinning are similar to AGA in young males. Males who experienced the first onset of scalp hair thinning after age 60 were compared to age-matched males (controls) without a history of hair thinning. Four scalp biopsies, two from the frontal and two from the occipital scalp, were obtained for horizontal sectioning and biochemical assay. Histologic findings were primarily follicular downsizing. Follicular drop out was not detected using elastic tissue staining, and there was no significant difference in number of follicles in frontal compared to occipital scalp. Senescent thinning was indistinguishable from androgenetic alopecia in older males. Inflammatory changes were not a significant feature. Biochemical analysis for androgen receptors, 5 -reductase type 1 and 2, and aromatase, in scalp biopsies from older males showed nearly a two fold decrease in levels compared to levels in young males with AGA. In males over 60, androgen receptor and aromatase levels were low and comparable in scalp with and without thinning in both frontal and occipital regions. The 5a -reductase type 1 and 2 levels were only slightly higher in males with thinning hair in both frontal and occipital regions, but the differences were not significant. Histologic and hormonal findings suggest that senescent thinning is a diffuse process that is histologically similar to AGA, but hormonally different and may not be entirely androgen dependent. We hypothesize that senescent alopecia is distinct from AGA.
High Efficacy Gene Therapy of Growing Hair Shafts.
AntiCancer, Inc., San Diego, CA 92111; 2 Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Japan.
A novel gene therapy technology of hair follicles has been developed which results for the first time in efficient genetic and phenotype alteration of the hair shaft. Mouse anagen skin fragments in histoculture were transduced at high efficiency by adenoviral-green fluorescent protein (GFP) with subsequent grafting of the skin fragments to nude mice. The histocultured skin fragments were treated with collagenase which made the hair follicles accessible to adenoviral-GFP enabling high-efficiency transduction. On day-1 after gene transduction, GFP was expressed in 80% of hair follicles in the collagenase-treated histocultured skin. GFP was visualized in the hair bulbs and dermal papillae in the histocultured skin. Without collagenase treatment, GFP expression was negligible. The extent of GFP expression in the hair follicle depended on adenoviral titer and duration of adenoviral treatment. High GFP expression was maintained for at least 18 days in histoculture. After transplantation of the GFP-transduced skin to nude mice, GFP was readily visualized in as many as 75% of hair follicles including large numbers of GFP-fluorescent growing hair shafts. Thus, transgene expression was maintained in vivo in hair follicles to the extent that growing hair shafts were transformed as demonstrated by their specific GFP fluorescence. RT-PCR confirmed GFP-gene transfer and expression in the hair follicle in histoculture as well as after grafting for at least 2 weeks. These experiments thus demonstrate that hair follicle gene targeting ex vivo enable for the first time high efficiency gene transduction which results in extensive hair shaft alteration in vivo. This novel technology indicates the possibility of efficient clinical genetic modification of the hair shaft such as during the hair transplant process.
TrichoScan: Combining Epiluminescence Microscopy with Digital Image Analysis for the Measurement of Hair Growth in vivo
Rolf Hoffmann, Dept. of Dermatology, Philipp University, Marburg, Germany
Numerous methods have been reported to assess the rate of hair growth, but most techniques are of little use to the clinician, because they are time consuming, costly or difficult to perform. Therefore, an operator- and patient-friendly, inexpensive, validated and reliable method is a rational need. Such a method must be able to analyze the biological parameters that constitute hair growth, which are: 1: hair density (n/cm 2), 2: hair diameter (µm), 3: hair growth rate (mm/day) and 4: anagen/telogen ratio. We have developed such a method which combines an epiluminescence microscopy with a newly programmed digital image analysis. The application of the technique is demonstrated in 30 volunteers (with or without AGA) where a scalp area of 1.8 cm 2 was clipped, dyed and land marked with a central, single black tattoo. Images were taken at 20 and 40-fold magnification immediately and 2 days after shaving, in three monthly periods. Our results show that the technique is able to automatically analyze the anagen/telogen ratio (< 18 % telogen hairs in scalp unaffected by AGA) and hair growth rate (mean 0.31 mm/day). We were able to demonstrate that within the very small analyzed area (0.225 cm 2) the total hair number and hair thickness at the occiput did not change during 6 months, but we observed a decrease in hair number (-0.43 hairs) and total thickness (-1.85µm) in AGA-affected untreated scalp in contrast to an increase in hair number (+ 4,7 hairs; p = 0.059) and total hair thickness (+4.85µm; p = 0.039) after six months of treatment with finasteride. The advantage of this technique is that it can be used for clinical studies to compare placebo versus treatment, or to compare different capacities of different hair growth-promoting substances. This technique can be used for studying AGA or other forms of alopecia, but in addition can be adopted to study the effect of drugs or laser treatment on hypertrichosis or hirsutism.
New developments in alopecia areata using rodent models
K.J. McElwee. Philipp University, Marburg, Germany.
Rodent models of human disease provide an important tool in the investigation of genetic and environmental activation factors, disease pathogenesis, and the development of new and improved treatments. Several inbred rodent models have been identified for alopecia areata (AA), a non-scarring inflammatory hair loss disease with suspected autoimmune elements. Up to 20% of C3H/HeJ mice and 70% of Dundee Experimental Bald Rats (DEBR) develop AA-like, patchy hair loss that may progress to universal alopecia. Hair loss is associated with peri- and intra-follicular lymphocyte and macrophage inflammation, but no significant scarring. These two rodent models have been employed in examining the mechanisms involved in AA. Manipulation of inflammatory cells by selective in vivo cell depletion or transfer between affected and unaffected C3H/HeJ mice indicates AA is primarily a cell mediated disease. Successful induction of AA by skin graft transfer between affected and unaffected mice has been used to examine changes in gene expression within AA-affected skin using flow cytometry and gene array technology. Rodents are now utilized to evaluate a variety of current treatments and for developing new therapies. The ability to follow the full course of AA throughout life including events prior to the onset of actual hair loss makes rodent models a considerable asset in AA research. The ability to induce AA-like disease with visible hair loss several weeks after surgery in the mouse AA model provides a consistent, controllable model for disease investigation and novel drug efficacy studies. Ultimately, animal models will be used to determine the genetic basis of AA, the potential endogenous and/or environmental trigger(s), the mechanism(s) of disease initiation and progression, and allow evaluation of new treatments.
Effect of Coapplication of Capsaicin and Minoxidil on the Murine Hair Growth
Won-Soo Lee, Hyung Jin Ahn, Young Hee Kim
Department of Dermatology. Yonsei University Wonju College of Medicine, Wonju. Korea
Capsaicin induces the release of substance P(SP) which is believed to play an important role in murine hair growth and cycle. Minoxidil, the only approved topical hair drug having direct hair growth effect, has therapeutic limitation because it stimulates mainly vellus hair regrowth, not thick coarse terminal hairs. So, we hypothesize that combined use of capsaicin and minoxidil may exert positive effects in terms of hair growth than minoxidil alone. We evaluated the effects of coapplication of capsaicin and minoxidil on the hair growth and cycle, compared with application of capsaicin or minoxidil alone. We induced the anagen phase of the hair of back skin of ICR mice by depilation. We divided the mice into 4 groups, i.e., control group, capsaicin group, minoxidil group, and coapplied group. And then, we examined the hair growth macroscopically, and the percentage of the area of hair regrowth by image analysis using phototrichogram at the 0, the 5th, 10th, 15th, 20th, 25th, and 30th day. Also we examined the morphologic changes of hair follicles and subcutis, and the number of mast cells by microscopy, andmethyl-3Hthymidine uptake by scintillation counter. In this study, we observed that capsaicin can not only induce the anagen phase quickly, but also sustain constant effect on the linear hair growth. Minoxidil also induced the anagen phase fast, and prolonged the anagen phase of the hair cycle. Therefore, it is concluded that the coapplication of capsaicin and minoxidil can grow hair quickly and steadily.
Alopecia areata: Treatment today and tomorrow
Rolf Hoffmann, Dept. of Dermatology, Philipp University, Marburg, Germany
Alopecia areata is a tissue-restricted, T-cell mediated autoimmune disease of the hair follicle. Current treatments for alopecia areata involve immunosuppression by corticosteroids or PUVA. So far, attempts to treat alopecia areata with immunophilin-ligands such as FK506 or ASM981 have not been succesful. The most effective treatment for severe alopecia areata is the use of contact sensitizers such as diphenylcyclopropenone and squaric acid dibutylester. Although their mode of action is not precisely known, there is evidence that they mediate their beneficial effect in alopecia areata via the induction of the immunosuppressive cytokines TGF-ß and IL-10. Dictated by the autoimmune pathogenesis of AA, improved future treatments may be immunosuppressive, immunomodulatory, or protect the hair follicle from the injurious effects of the inflammation. As disease onset inhibitors are unlikely to be developed in the foreseeable future, research into new treatments must first focus on disease symptoms. Such possible future treatments are discussed, including the use of cyclosporine- or FK506-loaded liposomes, application of immunosuppressive cytokines like TGF-ß and IL-10, inhibition of apoptosis mediated by the Fas-FasL system, inhibition of the lymphocyte homing receptors CD44v10 or CD44v3, induction of tolerance with tolerogenic dendritic cells and gene therapy.
Suppression of TGF-ß prevents apoptosis induction in the catagen hair follicle
MGH/Harvard Cutaneous Biology Research Center, Charlestown, MA, USA
Hair loss is the result of premature entry into catagen by various causes. In male pattern baldness, we have hypothesized the involvement of “catagen cascade”, in which a TGF-β family member promotes caspase activation, resulting in the apoptosis of epithelial cells. In the previous study, we showed that TGF-2 was localized at the boundary area between germinative cells and DPC during the transition phase from late anagen to catagen. In the epithelial strand TGF-β 2 was detected in the regressing hair follicle. TUNEL-positive apoptotic cells and active caspase–3 and –9 were also observed in the area. In this study, we examined the role of TGF-β 2 in relation to apoptosis. Human hair follicles were cultured in the presence of TGF-β 2. Using active caspase-9 and –3 specific antibodies, we found that TGF-β 2 enhances the activation of these caspases in two regions, including the lower part of germinative matrix cells and outer most layer of outer root sheath cells. Dual staining for active caspase-9 and TUNEL demonstrated that active caspase-9 and TUNEL-staining mostly co-localized. Active caspase-3 positive cells were detected in the same region. We evaluated 400 plant extracts for activity in a TGF- b suppression assay. An extract from Hydrangea macrophylla strongly inhibited TGF-β induction in this assay and also promoted hair elongation in a hair follicle organ culture system. We tested the effect of the extract in vivo by applying it onto C57BL6 mouse for 10 days and scored the catagen stage of each hair follicle morphologically. Topical application of the extract remarkably delayed the progression of catagen. We isolated an active substance from the extract using a TGF-β suppression assay. This substance showed a strong potential for hair elongation and also reduced caspase activation in cultured follicles. Collectively our results suggest that TGF-β 2 can enhance the induction of catagen via activation of caspases and that the suppressor of TGF-β could be effective for the prevention of male pattern baldness. We are presently investigating the role of TGF-β family members and their signal pathways in the endogenous mouse hair cycle.
Androgen-inducible TGF-beta1 Derived from Dermal Papilla Cells Mediates Hair Growth Suppression in Androgenetic Alopecia
Shigeki Inui, Yoko Fukuzato, Takeshi Nakajima, Kunihiko Yoshikawa, Satoshi Itami
Department of Dermatology, Osaka University Medical School
Although androgen plays the central role in androgenetic alopecia (AGA), the pathomechanism is unsettled at the present because of the absence of suitable in vitro model system. It has been reported that androgen inhibits the proliferation of outer root sheath cells cocultured with dermal papilla cells (DPCs) from the frontal bald scalp of stump tailed macaques, a model animal for human AGA, while this was not the case in human DPCs. In this study, we attempted establishing a coculture model of human bald DPCs and keratinocytes to identify the pathogenic mediators for the inhibition of epithelial cell growth in AGA. Since we found that the expression of mRNA of androgen receptor (AR) decreased during subcultivation of DPCs in vitro, we transiently transfected the AR expression vector into bald DPCs by lipofection and cocultured them with keratinocytes. In this coculture system, androgen suppressed the growth of keratinocytes by 50%, indicating that exogenous overexpression of AR can restore the phenotype of bald DPCs. The androgen-induced inhibition of keratinocyte proliferation was antagonized by the addition of cyproterone acetate (CA), a potent antiandrogen, to the coculture of keratinocytes and bald DPCs, suggesting the specific role of AR in DPCs for the growth inhibition of cocultured keratinocytes. We further examined the change of expression level of mRNA of various factors in bald DPCs cocultured with keratinocytes after androgen treatment by semiquantitative RT-PCR and found that TGF-ß1 was increased by androgen in bald DPCs. Moreover, the neutralizing anti-TGF-ß1 antibody antagonized the inhibition of keratinocyte proliferation in this co-culture system in a dose dependent manner. From these data obtained using our novel powerful coculture model for AGA, we suggest that androgen-inducible TGF-ß1 derived from DPCs mediates hair growth suppression in AGA.
Recapitulation of the hairless mouse phenotype using catalytic oligonucleotides.
Peter B. Cserhalmi-Friedman, Andrei A. Panteleyev, and Angela M. Christiano.
Departments of Dermatology and Genetics & Development, Columbia University New York, NY
Ribozyme technology is widely used to target mRNA in a sequence specific fashion, and thus change the expression pattern of cells or tissues. While the goal of mRNA targeting is usually the cleavage of mutant mRNA with the prospect of gene therapy for inherited diseases, in certain instances targeting of wild-type genes can be used therapeutically. Lack of expression of the mouse hairless gene due to inherited mutations leads to the complete loss of hair, known as atrichia. We designed this study to recapitulate the hairless phenotype in a restricted manner by topical application of deoxy-ribozyme targeting molecules to specifically cleave the mouse hairless mRNA. The targeting oligonucleotides were designed using previously reported catalytic core structure and the mRNA sequence of the mouse hairless gene. The targeting molecules were delivered on the back of C57Bl/6J mice using a commercially available liposome reagent. The delivery began immediately after birth, and was repeated daily until day 16. Patches of hair loss were grossly visible on treated areas of dorsal back. Samples for pathology were taken at days 22 and 35, processed and stained using standard techniques. The pathology samples of the treated area from day 22 demonstrated a decreased number of hair follicles, involution of the remaining follicles and separation of the dermal papillae, all characteristic of the hairless phenotype. In contrast, the pathology samples from the non-treated area of the same animal did not show these alterations. The pathology samples of the treated area from day 35 demonstrated the presence of large dermal cysts characteristic of the hairless phenotype, but not normally present in the skin of C57Bl/6J mice. In this study, we successfully recapitulated the hairless phenotype using topically applied target specific catalytic oligonucleotides designed to cleave the mouse hairless mRNA. Our results demonstrate the feasibility of using ribozyme technology to alter gene expression in the skin via topical application, and provide proof of principle for the development of this strategy for permanent hair removal.
Molecular control of chemotherapy-induced hair loss: essential roles for p53 and its target genes
Dept. of Dermatology, Boston University School of Medicine
Dept of Molecular Genetics, University of Illinois at Chicago
Dept. of Dermatology, University of Mainz, Germany
Anticancer drugs induce apoptosis in the hair follicles and hair loss, the most common side effect of chemotherapy. In C57BL/6 mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: (i) by immunohistology, p53 was upregulated in the hair follicles after cyclophosphamide treatment, (ii) in contrast to wild type mice, p53-deficient mice show neither hair loss nor apoptosis in the hair follicle keratinocytes that maintained active proliferation after cyclophosphamide treatment, (iii) hair follicles in p53 knockout mice are characterized by downregulation of Fas and insulin-like growth factor binding protein-3, and increased expression of Bcl-2. To confirm a role for Fas as a p53 target gene in chemotherapy-induced hair loss, we show that Fas knockout mice display significant (p<0.05) retardation of cyclophosphamide-induced hair follicle regression and decrease of intrafollicular apoptosis. Furthermore, administration of Fas-ligand neutralizing antibody significantly reduced a number of apoptotic cells in the hair follicle and retarded a rate of hair follicle involution induced by cyclophosphamide. These observations indicate that local pharmacological inhibition of p53 and/or its target genes may be useful to prevent chemotherapy-associated hair lossProlactin Receptor Knockout Mice Have Altered Hair Growth Cycles INSERM Unité 344, Paris, France.Although prolactin has been shown to entrain hair growth cycles in seasonally responsive mammals, no comparable role has been identified in the age-dependent pelage replacement of rodents. We therefore analysed hair growth cycles in prolactin receptor gene-disrupted mice (PRLR -/- ) to determine the influence of prolactin signaling on murine hair cycles. The second (G2) hair cycle of PRLR -/- mice on the 129SV background, and their wildtype (PRLR +/+ ) and heterozygote (PRLR +/- ) littermates, were visualized and compared by dyeing their coats at 28 days post-partum and observing the emergence of undyed fur. Samples of mature pelage were collected for measurement of fibre length and diameter distribution. The growth development status of hair follicles in adult and neonatal mice was determined by skin histology. The expression of PRLR in the skin was detected by RT-PCR and immunocytochemistry. The hair growth phenotype of PRLR -/- mice consisted of a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR -/- neonates, G2 hair replacement in PRLR -/- mice occurred earlier than in wildtypes. In female PRLR -/- mice, fibres erupted on the dorsum by 33.0 ± 0.7 days of age in mice in contrast to 61.9 ± 2.8 days in PRLR +/+ mice (P<0.001). PRLR + - mice were intermediate (50.1 ± 3.2 days). In males, a similar effect, but with a much reduced difference between the PRLR -/- and PRLR +/+ genotypes was observed (31.0 ± 1.0, and 34.9 ± 0.7 days respectively; P<0.001). Thus PRLR deficiency eliminated the sexual dimorphism associated with murine hair replacement. Once initiated, the pattern and progression of hair replacement across the body was similar in all genotypes. All fibre types were present and appeared structurally normal, but PRLR -/- mice had slightly longer (P<0.001), and coarser (P<0.05) hair than wildtypes. mRNA encoding both the long and short-3 forms of prolactin receptor were found in the skin of both adult and neonate wildtype mice. The receptor protein was immunolocalized to the outer root sheath of the hair follicle as well as the epidermis, sweat and sebaceous glands. These findings suggest that prolactin has an inhibitory effect on murine hair cycle events.Gene expression profile in dermal papilla cells and construction of hair specific cDNA microarrays Kyungpook National University, School of Medicine Department of Immunology Kim Moonkyu, Kim Young-Hee, Im Sang-Uk, Hwang Sun-Young, Chung Eun-Jung, Kim Do-Won, Kim Jung-ChulPartial sequencing of randomly selected clones from cDNA libraries of specific tissues or cell types to generate expressed sequence tags (ESTs) has proven to be a rapid and efficient means of discovering genes on a large scale and of providing both quantitative and qualitative information regarding gene expression in a variety of tissues. To gain an insight into the genetic profile of human dermal papilla cells, we have constructed cDNA library from cultured dermal papilla cells and generated an expressed sequence tag (EST) database from them. We constructed cDNA libraries from cultured human scalp hair dermal papilla cells. The 5’ ends of 1,666 randomly selected plasmid clones, each contains one gene which expressed in dermal papilla cell, were sequenced and searched for sequence homologies in the GenBank DNA data base by BLAST. It generated human dermal papilla cell EST database with an average length of 180 nucleotides. Sequences of 351 clones (21.1%) showed no significant similarity to entries in the public databases; 1,229 (73.8%) were identical to known human sequences. The most abundant mRNAs were for type 1(I) collagen chains and fibronectin (53 and 49 times, respectively). mRNAs for osteonectin, α 2(I) collagen, actin-beta, SM22-alpha, and glyceraldehyde-3- phosphate dehydrogenase. Clones for gamma-actin, α 1(VI) collagen, plasminogen activator inhibitor 1, IGH3, α 2(VI) collagen, and ferritin heavy chain showed some redundancy. Clones corresponding to a total of 1,229 specific known genes were classified according to their function. The most abundant class of genes expressed in dermal papilla, were those for cell structure and motility, which comprises 366 cDNA clones (29.8%), including type I collagen, type IV collagen, fibronectin, osteonectin, beta-actin, alpha-SM22, gamma-actin, and IGH3. All of the genes in human dermal papilla EST database are available through our web site at http://hair.knu.ac.kr. We also have constructed hair-specific cDNA microarrays containing 3,066 cDNAs using dermal papilla EST clones. Those dermal papilla cDNA collection and microarray can provide valuable information in the field of hair research.Factors that Mediate and Modulate Androgen Action MJ McPhaul and M Young, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TXAndrogens mediate a wide range of processes during embryogenesis and in the adult. In mammals, although a number of steroids can be shown to exert androgenic effects using in vitro and in vivo assays, testosterone and its 5 reduced metabolite, 5 -dihydrotestosterone (DHT) are considered to represent the principal androgens in mammals. Furthermore, although the effects that androgens exert differ widely between different tissues and cell types, genetic and biochemical data suggest that these effects are mediated via the protein products of a single androgen receptor gene, which is encoded on the X-chromosome. The last decade has witnessed an explosion of information regarding the manner in which steroid hormones modulate gene expression. At present, a body of increasingly detailed information is available as to the mechanisms by which nuclear receptors, such as the androgen receptor, regulate the activity of target genes. The studies have demonstrated the participation of a number of ancillary proteins in modulating the activation or repression by nuclear receptors. These proteins have been shown to possess a variety of activities, including the capacity to modify chromatin structure. In parallel to experiments focused on the mechanism by which nuclear receptors regulate the transcription of responsive genes, experiments have demonstrated the importance of androgen metabolism in specific cell types. A number of enzymes capable of catalyzing the inactivation or synthesis of active androgens have been described. The complement of activities expressed in an individual tissue or cell type is likely to affect the degree of androgen - responsiveness. The results of such investigations provide a perspective on the number of levels of complexity by which differential gene regulation by androgens may occur in different cell types. Such insights may provide avenues by which to modulate the actions of androgens selectively in specific tissues.Diagnosis and Treatment of Androgenetic Alopecia in Women V.H. Price, Dept. of Dermatology, University of California San Francisco, San Francisco, CA, USAAndrogenetic alopecia (AGA) is caused by androgens in genetically susceptible women and men. The thinning, also known in women as female pattern hair loss, begins between ages 12 and 40 years. Inheritance pattern is polygenic, and incidence is the same as in men. In susceptible hair follicles, dihydrotestosterone binds to androgen receptor, and the hormone-receptor complex activates the genes responsible for the gradual transformation of large terminal follicles to miniaturized follicles. As in young men with AGA, young women have more androgen receptors, higher levels of 5 alpha reductase and lower levels of aromatase, in frontal than in occipital follicles. However, the process is milder in women than in men because of differences in these levels. The miniaturized follicles produce shorter and finer hairs of various lengths and diameters, and these miniaturized hairs are the hallmark of the condition. In women, hair thinning is diffuse but most marked on the frontal and parietal scalp. Wo n typically retain a rim of hair along the frontal hairline. Increased spacing between hairs makes the central part look wider over the frontal scalp compared to the occipital scalp. Diagnosis is supported by these features including miniaturized hairs, pattern of thinning, and early age of onset. Most women with AGA have normal menses, pregnancies. Extensive hormonal testing is usually not needed unless symptoms and signs of androgen excess are present such as hirsutism, severe unresponsive cystic acne, virilization, or galactorrhea. Androgen excess is assessed by measurment of serum total or free testosterone, dehydroepiandrosterone sulfate, prolactin, and other causes of hair loss are ruled out by serum thyrotropin, iron and ferritin. Topical minoxidil solution is the only drug available for promoting hair growth in women with AGA. Efficacy has been shown in double-blind studies using hair counts and hair weight. Minoxidil solution must be applied twice daily directly to a dry scalp. Women are devastated by their thinning hair and need reassurance that they may safely use hair cosmetics to make their hair appear fuller.Endocrine controls of hair growth: New Developments Ralf Paus, Dept. of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, GermanyWhile the significance of androgens as major modulators of hair growth is well-established, much less is known on the exact follicular functions of other nuclear hormone receptor ligands (e.g. endogenous estrogens, thyroid hormones, retinoids, calcitriols, glucocorticoids) and on non-steroidal, peptide hormones and neuropeptides (e.g. ACTH, aMSH, CRH, prolactin, substance P, catecholamines, melatonin). This introductory synthesis begins by summarizing important relevant findings from the old endocrine literature (mostly in rodents) that serve to highlight many of the most intriguing open questions on the hormonal controls of hair growth, both in an endocrine d in a paracrine/autocrine signalling context, which the overly dominant androgenocentric view of the hair follicle has tended to obscure. This is followed by a discussion of more recent findings that document the hair follicle to be both a source and a target of numerous hormones, neuropeptides and neurotransmitters, whose expression (along with that of their cognate receptors) underlies tight, hair cycle-dependent regulatory controls. In particular, the hypotheses are explored that the hair follicle exploits several locally generated and/or metabolized hormones for controlling its own growth, innervation, immune functions, perfusion and/or pigmentation and that the hair follicle has established a miniature equivalent of the hypothalamic-pituitary-adrenal axis, possibly as part of a complex, local stress response-system.Role of Stat3 in hair development revealed by the conditional gene targeting Takeda J. Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine 2-2 Yamadaoka, Suita, Osaka 565-0871, JapanSignal transducer and activator of transcription 3 (Stat3) is involved in many signaling pathways for growth factors and cytokines. Its deficiency in mice therefore causes early embryonic lethality, preventing analysis of the functions of the Stat3 gene in adulthood. To circumvent this difficulty and explore the functions of Stat3 in the epidermis, the keratinocyte-specific conditional gene targeting using the Cre/loxP system was utilized. Despite functional ablation of Stat3 in the keratinocytes from the mutant mice, no developmental abnormality was observed. However, skin remodeling such as wound healing and second hair cycling was profoundly impaired in the mutant mice. In an in vitro culture system, the migration of keratinocytes from the mutant mice stimulated with various growth factors and cytokines was severely attenuated, which is thought to account for the defect of skin remodeling in the mutant mice. Although the molecular mechanism of Stat3 involvement in keratinocyte migration remains unknown, focal adhesion is enhanced in the mutant, suggesting that Stat3 might be involved in the detachment of keratinocytes from the extracellular matrix rather than the attachment to it. However, the migration by PKC signalling was intact in the mutant mice. These data suggest that Stat3 and PKC pathway were independent for the keratinocyte migration. Wortmannin, an inhibitor of PI3K, impaired the migration of both pathway, implicating PI3K is critically involved in the keratinocyte migration.Targeted Disruption of LIG-1 Gene Provides New Insight into Keratinocyte Stem Cells. Safety Research Laboratory, Tanabe Seiyaku Co. Ltd., Osaka, Japan.Epidermis that covers the skin surface is continuously regenerated throughout the lifetime of the mammalian adult through proliferation of keratinocyte stem cells. While keratinocyte stem cells play a central role in tissue homeostasis, wound healing, cancers, and skin-based gene therapy, the precise in vivo localization of the cells is not yet settled because of the scarcity of appropriate molecular markers. LIG-1, a transmembrane glycoprotein of which extracellular region is uniquely organized with the leucine-rich repeats and immunoglobulin-like domains, is expressed predominantly in the brain. Here we show that the cutaneous expression of LIG-1 was restricted to the cells in the bulge of hair follicles and the subpopulation of epidermal basal cells that were considered to be the keratinocyt stem cells in mice and human. About 40 % of α 6 -integrin positive rapidly adherent keratinocytes that were enriched in keratinocyte stem cells strongly expressed LIG-1 on their cell-surface. To clarify the physiological roles of LIG-1 in vivo, we disrupted the gene in mice by gene targeting. The LIG-1 deficient mice developed a skin change on their tail and facial area after birth. The affected skins were histologically characterized by epidermal hyperplasia, hyperkeratosis with parakeratosis, neutrophil influx, and the subcorneal pustules similar to Munro’s microabscesses. The keratinocytes in the lesion were highly proliferative with perturbed terminal differentiation. Therefore, we infer that LIG-1 is a new cell-surface marker for keratinocyte stem cells and regulates the growth and differentiation of keratinocyte stem cells.Minoxidil stimulated hair growth in organ culture is inhibited by the potassium channel blocker, tolbutamide. GC Davies, TJ Jenner, YC Chen, VA Randall, MJ Thornton.Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, UK. Although potassium channel openers such as minoxidil promote hair growth in vivo, the mechanisms by which they do so are unclear. Using a red deer whole hair follicle culture system we have previously demonstrated that the potassium chan l openers, minoxidil and diazoxide stimulate hair follicle growth in vitro. In order to further explore the role of potassium channels in hair growth, we have investigated the effect of the potassium channel blocker, tolbutamide on red deer hair growth in organ culture. Anagen follicles were microdissected from the mane of 6 red deer stags, washed with sterile phosphate buffered saline, and incubated in Williams E medium supplemented with 5 mM glucose, 100 U/ml penicillin and 2.5 µg/ml amphoterin B. A minimum of six follicles were incubated in each experimental group; control vehicle (0.01 % dimethylsulphoxide), 0.1, 1, 10, 100 µM minoxidil, tolbutamide (1 mM), or minoxidil (10 µM) + tolbutamide (1 mM). Hair follicles grew under all experimental conditions for up to 8 days. Follicles consistently grew faster in the presence of ll concentrations of minoxidil compared to control conditions. In contrast, tolbutamide slowed follicle growth compared to the vehicle control. Incubation of isolated follicles with a combination of minoxidil and tolbutamide resulted in an inhibition of the stimulatory effects of minoxidil; the follicles grew at the same rate as the control follicles. The stimulatory effect of minoxidil at 4 different concentrations supports our earlier studies using this culture system. The absence of any growth potentiation by minoxidil in the presence of the potassium blocker, tolbutamide, implies that minoxidil is stimulating hair growth in this system via potassium channels. Further studies should help us to elucidate the exact mechanisms involved in the stimulation hair growth by minoxidl.Nitric Oxide Production of Human Dermal Papilla Cells: Basal and Androgen stimulated Expression of Constitutive and Inducible Nitric Oxide Synthase Inst. of Pharmacology, University Medical Center Benjamin Franklin, The Free University Berlin, Berlin,GermanyNitric oxide (NO) has been identified as an important mediator in various physiological and pathophysiological processes of the skin, such as regulation of blood flow, melanogenesis, wound healing, and hyperproliferative skin diseases. However, little is known about the role of NO in the human hair follicle and in hair cycling processes. By measuring nitrate and nitrite levels in culture supernatants we demonstrate that dermal papilla cells (DPC) derived from human hair follicles spontaneously produce NO. This biomolecule is apparently formed by the endothelial isoform of nitric oxide synthase (NOS), which could be detected by reverse transcription polymerase chain reaction (RT-PCR) and immunological protein analysis. Remarkably, basal NO level was markedly enhanced by stimulating DPC with 5 -dihydrotestosterone (DHT) but not by testosterone. Addition of N-(3-(aminomethyl)benzyl-acetamidine (1400W), a highly selective inhibitor of human inducible NOS, restrained the elevation of NO level induced by DHT. Analysis of androgen-stimulated cells on RNA as well as on protein level, respectively, confirmed the expression of inducible NOS. Thus, we suggest that endothelial NOS accounts as a constitutive enzyme for a physiological role of NO in hair follicle cells, whereas, upregulation of NO production by androgens via stimulating inducible NOS expression may indicate an involvement of NO in the pathogenesis of androgen-dependent hair loss, such as androgenetic alopecia. These findings indicate that NO could be part of an important signaling pathway in the human hair follicle.A Significant Role of Aromatase Cytochrome P450 on Hair Re-growth and Pigmentation.It is well known that sex hormones such as androgen and estrogen influence the hair growth in several animals. Aromatase is a member of cytochrome P-450 gene superfamily and catalyzes the conversion of androgen to estrogen, a rate-limiting step of estrogen biosynthesis. Previous reports have suggested that aromatase exists in various extra-gonadal tissue as well as in gonadal tissue, and that the expression is regulated by alternative use of multiple exons 1 and promoters. In this study, in order to examine the involvement of aromatase in hair growth, we investigated the localization of aromatase at hair follicles in human and murine, and evaluated the role of this enzyme on hair re-growth using aromatase knockout mice. Immunohistological technique for anagen hair follicles revealed that aromatase positive cells localized in outer root sheath tissue while were not observed in hair matrix, follicular papilla and epidermis. RT-PCR analysis of aromatase mRNA in plucked human hair suggested that aromatase transcripts were mainly composed of placenta-specific exon 1. Next, we examined the effect of aromatase inhibitor on the hair re-growth of C3H mouse. e topical application of 1% aminoglutethimide significantly promotes the hair re-growth. Moreover, hair re-growth was significantly accelerated in aromatase knockout female mice in compared with wild type. Intriguingly, the color of re-grown hair of those mice was blacker than wild type and heterozygotes. Alteration of hair color was caused by the increase of melanin contents (total melanin and eu-melanin). These results suggest that aromatase plays an important role on hair growth and melanin synthesis in hair follicles through the local synthesis of estrogen.The Molecular Basis of Tooth Development and Its Implications in Signaling Systems in Hair Development Ichiro Satokata Div. of Developmental Biology, Dept. of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, JapanEpithelial-mesenchymal interactions govern the initiation of organ development, subsequent morphogenesis and terminal cytodifferentiation. The developing tooth of the mouse is a good model for studying the molecular basis of the signaling systems in epithelial-mesenchymal interactions. Recently, a number of genes which encode growth factors, transcription factors and cell surface molecules have been identified that play a role for tooth development and parts of the molecular details of signaling systems have been elucidated, particularly in the signal families BMP, FGF, Shh and Wnt. Antagonistic signaling of FGF8 and BMP2, -4 is suggested to determine the sites of dentition by restricting the expression sites of a paired box gene Pax9. Shh and a homeobox gene Pitx2 are also candidates for determinants of dentition sites. Subsequently, the four epithelial signal families induce differential activation of key transcription factors, resulting in determination of tooth identity and morphogenesis. FGF8 induces homeobox genes Barx1, Lhx6, -7, Msx1, Dlx1, -2, and a member of TGF-β superfamily ActivinbA. FGF4 induces Msx1. BMP2, -4 induce Msx1, -2, Dlx2, a HMG-box gene Lef1 and inhibit Barx1.Wnts also induce Lef1. Shh induces zinc-finger genes Gli1, -2, -3. By the analyses of gene knockout mice, several of these genes have been shown to perform essential functions in determination of tooth identity and morphogenesis. The mesenchymal expression of Msx1 which is induced by the epithelial signals of BMP2, -4 at the lamina stage induces mesenchymal expression of Bmp4 at the bud stage, resulting in formation of a positive feedback loop in the mesenchyme. The mesenchymal BMP4 signaling in turn induces the expression of Msx2, Lef1 and a cyclin-dependent kinase inhibitor p21 in the enamel knot of the epithelium. BMP2, -4, Msx2 and p21 in the enamel knot inhibit epithelial cell proliferation and induce apoptosis, whereas FGF4, -9 in the enamel knot stimulate cell proliferation of both epithelium and mesenchyme. Such an antagonistic signaling of BMP and FGF in the enamel knot is thought to regulate the balance between cell proliferation and apoptosis during tooth morphogenesis. Synergistic and antagonistic effects of signaling molecules and transcription factors are reciprocally and recursively used between epithelium and mesenchyme during tooth development. As the several studies have shown that genes which regulate tooth development are also required for development of other ectodermal organs such as hair follicle and mammary gland, a better understanding of the molecular basis of tooth development y provide an important clue to elucidate the signaling systems in hair development.Finasteride for androgenetic alopecia: A review of the clinical trials Jerry Shapiro, Division of Dermatology, University of British Columbia, Vancouver, CanadaDrake et al showed that median scalp and serum DHT levels decreased with 0.2 mg, 1 mg and 5 mg of finasteride after 42 days of treatment. Roberts et al confirmed that finasteride 1 mg daily was the optimal dose, with 1 mg and 5 mg superior to lower doses such as 0.2 mg/day. The 5 mg dose was not more efficacious than the 1 mg dose. Three double-blind, randomized, placebo-controlled studies were conducted in 1879 men ages 18 to 41 years with mild to moderate hair loss. Two of the studies enrolled men with vertex hair loss and one with frontal hair loss. Finasteride 1 mg tablets or placebo tablets were taken once daily for 24 months in the vertex studies and 12 months in the frontal study. All three studies showed a significant hair count increase in men treated with finasteride, with a significant decrease in hair counts in men treated with placebo. Finasteride stabilized hair loss in 83% of the cases with vertex hair loss atafter two years. and in 70% of cases for frontal hair loss atafter one year. The chances of mild to moderate visible regrowth are 661% on the vertex atfter 2 years and 37% on the frontal area after one year. Van Neste et al. using the phototrichogram method provided direct evidence that finasteride 1 mg daily promotes the conversion of hairs into the anagen phase. A study of finasteride in 136 post-menopausal women with androgenetic alopecia (AGA) showed no benefit compared with placebo. Finasteride is well tolerated and side effects are rare. Side effe ts include 1.8% decreased libido (1.3% placebo), 1.3 % erectile dysfunction (0.7% placebo) and 0.8% decreased ejaculate volume (0.4% placebo). There was no significant difference from the placebo group for each of these side effects taken alone, but there is a statistical difference when all side effects are considered together (3.8% vs 2.1%). Side effects will subside spontaneously in 58% most of those who decide to continue the treatment and are reversible upon cessation of treatment within a days or week.Hair Follicle Stem Cells: Past, Present, and Future. New York Univ. Med. Sch., New York, NY.The issue of keratinocyte stem cells is of central importance in epidermal homeostasis, wound repair, and carcinogenesis. One of the most accepted means to identify keratinocyte stem cells takes advantage of the fact that they are normally rarely cycling in vivo, and can be detected experimentally as the “label-retaining cells” (LRCs). When this approach was used to localize the rarely cycling cells of the hair follicle, all of the follicular LRCs were exclusively confined to the bulge – the part of the outer root sheath marking the lowest point of the upper, permanent portion of the follicle. Although it was customarily thought that the hair follicle and the epidermis were governed by separate populations of stem cells, it was puzzling that (1) very few such LRCs were found in the interfollicular epidermis and (2) interfollicular human epidermal cells had less in vitro proliferative potential than the upper follicular epithelial cells. We showed recently that the bulge stem cells give rise not only to the lower follicle, but also to young transit amplifying cells that migrate into normal newborn mouse epidermis as well as wounded adult mouse skin. This provides the first evidence that that the bulge represents a major repository of skin keratinocyte stem cells that may be bipotent as they can give rise to not only the hair follicle, but also to the epidermis. This finding has major implications on hair biology, the long-term maintenance of the epidermis, the pathogenesis of skin carcinomas, and the mechanism and regulation of skin wound repair.Growth Factor Secretion by Dermal Papilla Cells is Regulated by Neurotrophins.Neurotrophins have several non-neuronal functions in the skin. Previously we have shown that p75 kD neurotrophin receptor plays an important role during HF morphogenesis and that neurotrophins (NT) accelerate hair follicle (HF) regression (catagen), most likely by stimulating keratinocyte apoptosis via binding to the p75 kD neurotrophin receptor. In addition, dermal papilla (DP) cells express high affinity NT receptors TrkB and TrkC during the HF anagen-catagen transition. Given that the DP is inductive during hair follicle morphogenesis and cycling, NT may contribute to the regulation of this mesenchymal cell population by modulating their growth factor secretion. To test this hypothesis, matched cultures of human scalp DP cells and dermal fibroblasts (n=6) were grown in the presence of 0.5-50 ng/ml of NGF, BDNF, NT-3 or NT-4. Secretion of different growth factors (SCF, VEGF, TGFb2, KGF) known to be produced by DP cells during anagen was assessed after 48 h by ELISA. Both DP cells and dermal fibroblasts were found to express TrkA, TrkC, and p75 kD neurotrophin receptors by immunohistochemistry. TrkB receptors however, were detected on DP cells but not on dermal fibroblasts. The NT tested had no significant effect on DP cell or dermal fibroblast proliferation. By ELISA, secretion of SCF by DP cells into supernatants was significantly down regulated by BDNF and NT-3, while VEGF secretion was decreased after NT-4 treatment. BDNF also significantly decreased TGF 2 secretion by DP cells. Interestingly, the secretion of these growth factors by dermal fibroblasts was not affected by NT, suggesting that in addition to TrkB expression on DP, but not dermal fibroblasts, this secretary response to NT may aid the discrimination of interfollicular dermal fibroblasts from DP fibroblasts. These data suggest that NT may modulate HF cycling by altering secretion of DP-derived growth factors that control proliferation, apoptosis and/or differentiation of hair matrix cells.Signal Transduction Pathways and Cytokines Involved in Restoration of Hair Growth with Topical Anthralin in Alopecia Areata Rats. Liren Tang, Liping Cao, Steven Pelech, Harevey Lui, David I. McLean and Jerry Shapiro; Department of Medicine, University of British Columbia and Vancouver General Hospital, Vancouver, CanadaWe have shown that anthralin is very effective on hair restoration in the Dundee experimental bald rats (DEBR). All 15 rats showed near complete hair regrowth on the treated sides, while the control sides remained balding. The aim of the present study was to investigate the underlying molecular mechanisms of the therapeutic effects of anthralin on AA-affected rats. Skin biopsies were collected from both treated and control sides. RNA and proteins were extracted. Proteins were used for Kinetwork TM analytical screens for cell signaling proteins (Kinexus, Canada) to determine the expression of various protein kinases, phosphatases, and their down stream trascriptional regulators which might be responsible for the signal transduction pathways mediated by anthralin in rat skins. RNA protection assay was performed to determine the gene expression of various cytokines mediated by anthralin. Among the cytokines we have tested by RNA protection assay (RPA), interleukin 1b (IL-1 ), IL-1 , IL-10 and IL-1 receptor antagonist (IL-1Ra) showed remarkable induction after anthralin treatment, while tumor necrosis factor α (TNF-α ) and interferon γ (INF-γ ) were decreased upon successful treatment with anthralin. Protein kinase screening revealed that growth factor mediated signal transduction involving Erk1 & 2, their upstream regulators (Raf, Mek 1 & 2) and downstream kinases (p70 S6K and p90 S6K ), are all activated. The stress-activated pathways involving SAPK 1 & 2 and their upstream regulatory kinases (Mek 4 & 7) were also induced by anthralin, presumably owing to the free radicals generated by anthralin on the skin. In addition, another pathway mediated by PKB and GSK-3 kinases were also activated. Cytokine-mediated signals transduction pathways through Jak kinases were only weakly detected. But the downstream STAT-3 showed dramatic increase in its phosphorylation. We conclude that the molecular mechanism underlying the efficacy of anthralin on hair regrowth in AA rats might be mediated by the interplay of cytokines produced locally in the skin. The signal transduction pathways involving various protein kinases and their regulators may represent the initial molecular events in rat skin upon successful anthralin treatment. This line of information will help our understanding of the molecular mechanisms of anthralin’s efficacy on AA rats.
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