Peptidylarginine deiminase isoforms are differentially expressed in the anagen hair follicles and other human skin appendages

Peptidylarginine deiminases (PAD) catalyze the conversion of arginine residues to citrullines. Five isoforms are known that present distinct tissue locations. In the epidermis, like in the skin, only PAD1, 2, and 3 are expressed. Their pattern of expression in skin appendages is not known. Here, confocal microscopy analysis using highly specific antibodies demonstrated that PAD1 and 3 are expressed in human anagen hair follicles, PAD1 and 2, in arrector pili muscles and sweat glands, whereas no PAD were detected in sebaceous glands. PAD1 was detected in the cuticle and the Huxley layer of the inner root sheath (IRS), and in the companion layer. PAD3 was localized in the medulla, and in the three layers of the IRS. Using anti-modified citrulline antibodies, we also showed that deiminated proteins appeared in the lower part of the IRS, first in the Henle layer, then in the cuticle, and finally in the Huxley layer. Our data demonstrate that PAD3 is the enzyme that deiminates trichohyalin in the medulla and the Henle layer, indicate that PAD1 and 3 are involved in the hair follicle program of differentiation, and suggest a role for PAD1 and 2 in the physiology of sweat glands and arrector pili muscles.     

In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture

Successful hair follicle organ culture has been established for some time, but hair growth in vitro is limited and generally terminates prematurely in comparison with in vivo. The reasons why growth stops in culture are as yet unknown. In this investigation, adult rat vibrissa follicles for which growth in culture is limited to about 10 d, were maintained in vitro for a minimum of 20 d after the hair shaft stopped growing. The pattern of fiber growth and long-term follicle pathology reflected the initial hair cycle stage at the time of isolation. Furthermore, there was evidence that a group of follicles put into culture when in late anagen were attempting to cycle in vitro. Microscopy showed that, in spite of widespread pathologic changes to the follicle epithelium, dermal cells in the follicle showed remarkable resilience. Their viability was confirmed when primary cell cultures were established from isolated dermal tissue. These cells labeled positively for alpha-smooth muscle actin, an established marker of hair follicle dermal cell phenotype in vitro. Moreover, isolated dermal tissue induced hair growth when implanted into inactivated hair follicles in vivo. These data confirm that the cessation in hair growth is not due to a loss of the inductive capacity in the dermal component. Long-term organ culture may provide opportunities to investigate factors that are expressed or lost during hair growth cessation. In addition it may be possible to develop this method further to obtain a reliable and predictable model of hair follicle cycling in vitro.     

Overexpression of skin protein kinase C-alpha in anagen hair follicles during induced growth of mouse hair

A role for protein kinase C (PKC)-alpha has been implicated in the growth of mouse hair. Topical application of PKC activators, hair plucking, allergic contact dermatitis and skin irritation can all enhance growth of mouse hair, and a significant increase in PKC-alpha level in whole mouse skin in mature anagen has been demonstrated in these processes. Overexpression of PKC-alpha in anagen hair follicles has also been reported in natural growth of mouse hair. It is known that overexpression of PKC-alpha is associated with the acceleration of cell growth. Therefore, we postulated that overexpression of PKC-alpha in mature anagen may relate to enhancement of hair growth. The distribution of PKC-alpha in hair follicles during induced growth of mouse hair has not previously been studied. In this study, hair growth in C57BL/6 mice was induced by plucking the telogen hairs on one side of the back. The undepilated contralateral side served as a control. Expression of PKC-alpha in hair follicles during the hair growth cycle induced was evaluated by immunohistochemistry using cryosections and a specific polyclonal anti-PKC-alpha immunoglobulin G (IgG) antibody. No PKC-alpha was detected in telogen hair follicles or in the hair follicles at 1 day post-depilation, when the induced hair cycle was in early anagen. At 4 days after plucking, when the induced hair cycle was in mid-anagen, intense staining for PKC-alpha was found in hair papillae. At 10 and 17 days after depilation, when the induced hair cycle was in mature anagen and early catagen, respectively, all outer root sheath (ORS) cells and outer connective sheaths of hair follicles were stained positive. Because no PKC-alpha was detected in telogen hair follicles in this study, down-regulation of PKC-alpha in early anagen could not be observed. However, consistent with our previous findings, overexpression of PKC-alpha was found in mid-anagen and mature anagen. As overexpression of PKC-alpha has been shown to be associated with acceleration of cell growth, our results support the notion that PKC-alpha may play an important role in growth of hair follicle cells in induced growth of hair. As PKC levels are known to increase in hyperglycaemia, overexpressed PKC-alpha in mature anagen hair follicles may be related to the putative function of the ORS in mobilizing glycogen stores for anagen growth.     

Mimicking hair disorders by genetic manipulation of organ-cultured human hair follicles

Human hair follicles can be dissected out of scalp skin and cultured in vitro in defined growth medium. Hair follicle organ cultures have previously been used to investigate the molecular and cellular mechanisms through which various factors regulate the maintenance and cycling of adult hair follicles. In this issue, Samuelov et al. transfected organ-cultured human hair follicles with siRNA nucleotides and suppressed the expression of the endogenous P-cadherin gene in follicular keratinocytes. Knocking down the expression of P-cadherin in hair follicles in vitro recapitulated the hair follicle phenotype observed in patients with hypotrichosis with juvenile macular dystrophy (HJMD) and enabled the authors to establish a cause-effect relationship between loss of P-cadherin and suppression of the canonical Wnt signaling pathway and upregulation of TGFβ2 during development of the hair abnormalities observed in HJMD patients.     

Intermediate hair follicles: a new more clinically relevant model for hair growth investigations

Background Alopecia causes widespread psychological distress, but is relatively poorly controlled. The development of new treatments is hampered by the lack of suitable human hair follicle models. Although intermediate and vellus hair follicles are the main clinical targets for pharmacological therapy, terminal hair follicles are more frequently studied as smaller hair follicles are more difficult to obtain. Objectives This investigation was designed to quantify in vivo morphological and in vitro behavioural differences in organ culture between matched intermediate and terminal hair follicles, in order to develop a new clinically relevant model system. Methods Microdissected terminal and intermediate hair follicles, from the same individuals, were analysed morphometrically (250 follicles; five individuals), or observed and measured over 9 days of organ culture (210 follicles; six individuals). Results Intermediate hair follicles were less pigmented and smaller, penetrating less below the skin surface (mean ± SEM) (2·59 ± 0·07 vs. 3·52 ± 0·10 mm; P = 0·02), with smaller fibre (0·03 ± 0·002 vs. 0·07 ± 0·002 mm), connective tissue sheath (0·24 ± 0·01 mm vs. 0·33 ± 0·01 mm), bulb (0·19 ± 0·01 vs. 0·31 ± 0·01 mm) and dermal papilla (0·06 ± 0·002 vs. 0·12 ± 0·01 mm) diameters (P < 0·001). Intermediate hair follicle bulbs appeared ‘tubular’, unlike their ‘bulbous’ terminal follicle counterparts. In organ culture they also grew more slowly (0·044 ± 0·002 vs. 0·067 ± 0·003 mm per day; P < 0·001), remained in anagen longer (84 ± 0·03% vs. 74 ± 0·03% at day 9; P = 0·012) and produced less hair fibre (0·36 ± 0·02 vs. 0·50 ± 0·03 mm; P < 0·001) than terminal follicles. Conclusions Smaller intermediate hair follicles showed major morphological differences from terminal follicles in vivo and retained significant, biologically relevant differences in vitro in organ culture. Therefore, intermediate hair follicles offer a novel, exciting, more clinically relevant, albeit technically difficult, model for future investigations into hair growth. This should be particularly important for developing new therapies.     

Organ culture of human scalp hair follicles: effect of testosterone and oestrogen on hair growth

Summary Whole human scalp hair follicles were cultured. The follicles were dissected from skin pieces of normal scalp and put into 1.5 ml of incubation medium in a closed 5 ml glass tube under an atmosphere of 95% O₂ and 5% CO₂. The tube was rolled at 15 rpm at 36C. Remarkable hair growth was noticed for 7 to 8 days. Hair root sheaths also grew with the hair shafts. The structure of the hair bulbs was well maintained for at least 6 days, and then the hair matrix cells started to degenerate. Fetal calf serum was not essential for hair growth in vitro, but increased the growth rate slowly. Testosterone and oestrogen inhibited hair growth in vitro to a similar extent. The minimum effective doses of both hormones to suppress hair growth were around 5 ng/ml, which corresponds well to the normal plasma level of testosterone in adult males in vivo, suggesting that scalp hair growth may be critically controlled by testosterone in adult males.     

层黏连蛋白、纤连蛋白在人毛囊生长周期中的表达

目的 探讨层黏连蛋白(LM)、纤连蛋白(FN)在毛囊生长周期中的作用.方法 免疫组化S-P法检测LM、FN在毛囊生长期、退行期、休止期的表达.结果 ①生长期毛囊:LM均匀的表达于毛乳头处,在基底膜处呈明显的线状表达,外根鞘处表达也呈明显的阳性;FN均匀表达于毛乳头、基底膜、真皮鞘.②退行期毛囊:LM在毛乳头处少量表达,在基底膜处呈线状表达;FN在毛乳头和基底膜处表达均较生长期明显减少,但仍为阳性.③休止期毛囊:毛乳头浓缩成球点状,LM在毛乳头的表达为阴性,但在基底膜处仍然呈明显的线状表达,且较退行期更明显;FN在毛乳头、真皮鞘、基底膜处表达均为阴性.结论 LM、FN在毛囊的生长期、退行期及休止期的表达随毛囊生长周期而发生周期性变化,LM、FN可能参与毛囊生长周期的调节.     

胸腺素促毛囊生长作用研究进展

胸腺素是一类具有多重生物学功能的小分子多肽，普遍存在于各种组织细胞中，在生理和病理活动中起重要作用。胸腺素p4可通过激活毛囊干细胞、促进基质金属蛋白酶-2的合成和分泌及诱导血管生成等方式促进毛囊生长。通过开展相关的基础与临床研究，胸腺素类药物很有希望引入到一些顽固的脱发疾病的治疗中。     

Extracellular histones inhibit hair shaft elongation in cultured human hair follicles and promote regression of hair follicles in mice

Release of histone H4 in rat vibrissa dermal papilla (DP) cells exposed to sub‐toxic dose of colchicines has been recently reported. In addition, exposure to histone H4 has been reported to result in inhibited proliferation and reduced alkaline phosphatase (ALP) activity of cultured vibrissa DP cells. These findings prompted us to investigate the role of extracellular histones in hair growth using cultured human hair follicles and hair cycling using back skin of mice. We report here that exposure of cultured hair follicles to histone H4 and H2A resulted in significant inhibition of elongation of hair shafts, decreased expression of IGF‐1 and decreased expression and activity of ALP. Injection of histones into hypodermis of mice during anagen resulted in premature onset of catagen. Findings of the current study provide strong evidence suggesting the inhibitory role of extracellular histones in hair growth.     

Morphometry of human terminal and vellus hair follicles

Previous studies suggest that drug delivery systems based on particles can be used to deposit active compounds in hair follicles and to target hair follicle-associated cell populations. The development of application protocols is complicated by the fact that there is no information available on the size and the position of key target structures in the different hair follicle types and their intra- and interindividual variation. Therefore, we performed morphometric measurements on histological sections of human terminal (THF) and vellus hair follicles (VHF) from the scalp and the retroauricular region. With 3864 +/- 605 microm and 580 +/- 84 microm in THF compared to 646 +/- 140 microm and 225 +/- 34 microm in VHF, the total length and the length of the infundibulum differed significantly as determined by paired t-test (P < 0.0001). The same level of significance was observed for the position and the length of the bulge region. The thickness of the epithelial lining was lowest in VHF (45 +/- 14 microm at 100 microm from skin surface) compared to 65 +/- 20 microm at 150 microm in THF, while the thickness of the interfollicular epidermis ranged between 64 +/- 12 microm and 99 +/- 18 microm in VHF-bearing skin and 72 +/- 16 microm and 136 +/- 37 microm in THF-bearing skin. In addition, the diameter of the hair follicle opening was determined at 50 microm intervals from the skin surface. Our data suggest that hair follicle types in defined body regions represent rather homogenous groups and that particle-based drug delivery may be a feasible approach, also in larger numbers of individuals. We provide precise information on the size and the position of key target structures in VHF and THF.     

Co-culture of human hair follicles and dermal papillae in a collagen matrix

Human hair follicles, either alone or in combination with dermal papillae, were cultured in a collagen matrix. When plucked hair follicles were cultured alone, spike-like structures composed of outer root sheath cells started growing around the follicle and then radiated into the gel. When isolated dermal papillae were embedded close to the follicles, spikes started growing earlier and grew more rapidly than without the papillae. In cultures of excised follicles from which the dermal papilla had been removed, epithelial cells (possibly hair bulb cells) started growing out from the bulbous portion and then also formed spikes. In the presence of a papilla, the spikes elongated toward the papilla, finally reaching and surrounding it. These findings suggest that dermal papilla cells produce a factor(s) that enhances growth of follicular epithelial cells and also attracts those cells. In cultures of whole excised follicles, two major characteristic patterns of cellular growth were recognized. When the dermal papilla remained inside the bulb in contact with the hair bulb matrix, the hair matrix cells proliferated and differentiated in the normal manner, resulting in elongation of the hair shaft and follicle. But when the papilla was detached from the hair bulb matrix, epithelial cells proliferated from the bulbous portion and finally formed hair follicle-like structures. Thus, attachment of the dermal papilla to the hair bulb matrix in the bulbous portion appears to be necessary for growth of the hair and follicle in the normal manner. Our model may be useful for examining the interaction between follicular epithelial cells and dermal papillae and for studying the growth of hair and follicles in vitro.     

黄芩苷对人毛囊生长和毛乳头细胞分泌血管内皮生长因子的影响

目的 探讨黄芩苷对体外培养的人毛囊生长、毛乳头细胞增殖和分泌血管内皮生长因子(VEGF)的影响.方法 选择不同浓度黄芩苷作用于体外培养的人头皮毛囊8天,观察人毛囊生长及形态变化:作用于人毛乳头细胞72h,四甲基偶氮唑监(MTF)法测定细胞增殖活性,ELISA法检测细胞分泌的VEGF水平.结果 7.5.15,30,45μg/mL的黄芩苷对体外培养的人头皮毛囊有明显促生长作用;3.75,7.5,15,30μg/mL的黄芩苷对毛乳头细胞增殖无明显影响,但促进细胞分泌VEGF,与阴性对照组相比,差异有统计学意义.结论 黄芩苷促进体外培养的人头皮毛囊生长,部分可能是通过增加毛乳头细胞分泌VEGF促使毛发生长.     

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture

Summary In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-l3-acetatc (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (lC₅₀=1 nm). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 n m TPA. after which growth of TPA-treated follicles ceased while control follicles grew by a further 0–8 mm over the subsequent 6 days. In contrast. 10 n m TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0–79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of ³H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.     

Human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture

Of the numerous assays used to assess hair growth, hair follicle organ culture model is one of the most popular and powerful in vitro systems. Changes in hair growth are commonly employed as a measurement of follicular activity. Hair cycle stage of mouse vibrissa follicles in vivo is known to determine subsequent hair growth and follicle behavior in vitro and it is recommended that follicles be taken at precisely the same cyclic stage. This study was performed to evaluate whether categorization of human hair follicles by the growth in vivo could be used to select follicles of the defined anagen stage for more consistent culture. Occipital scalp samples were obtained from three subjects, 2 weeks later after hair bleaching. Hair growth and follicle length of isolated anagen VI follicles were measured under a videomicroscope. Follicles were categorized into four groups according to hair growth and some were cultured ex vivo for 6 days. Follicles showed considerable variations with respect to hair growth and follicle length; however, these two variables were relatively well correlated. Hair growth in culture was closely related with hair growth rate in vivo. Moreover, minoxidil uniquely demonstrated a significant increase of hair growth in categorized hair follicles assumed at a similar early anagen VI stage of hair cycle. Selection of follicles at a defined stage based on hair-growth rate would permit a more reliable outcome in human hair follicle organ culture.Oh Sang Kwon and Jun Kyu Oh contributed equally.     

Metabolism of foreign substances in human skin and hair follicles

Human skin has an inducible aryl hydrocarbon hydroxylase (AHH) activity which is dependent on cytochrome P-450. The cutaneous AHH-activity is inhibited by 7,8 benzoflavone but not by metapyrone, whereas recently published data showed that AHH-activity in human liver is enhanced by 7,8 benzoflavone and inhibited by metapyrone. These results suggest that AHH activity of human skin depends on different isoenzymes compared with those in human liver.