Expression and location of phospho-Artemis (Serine516) in hair follicles during induced growth of mouse hair

Artemis has been implicated in having a role in NHEJ, and it is also a multifunctional protein. Previous studies have found Omenn syndrome-like phenotype due to Artemis mutations and associated with alopecia. As Artemis phosphorylation in its c-terminus including Serine516 is prerequisite for the Artemis endonuclease reaction, we postulate that Artemis (Serine516) may be expressed in hair follicle and relate to hair cycling. In this study, hair growth in C57BL/6 mice was induced by plucking the telogen hair on the back. Expression of Artemis (Serine516) in hair follicles during the hair growth cycle was evaluated by immunofluorescence using cryosections and a specific polyclonal anti-Artemis (Serine516) immunoglobulin G (IgG) antibody. It was detected in germ cells, cap, and club hair adjoining the epidermis in telogen. In anagen II, intense staining for Artemis (Serine516) was found in the whole interfollicular epidermis, and in strand keratinocytes. In anagen IV, intense staining for Artemis (Serine516) was detected in basal cells and upper of outer root sheath (ORS) and inner root sheath (IRS). But only upper ORS and lower medulla were stained positive in anagen VI. Upper ORS and lower cortex were positively stained with Artemis (Serine516) in catagen. Based on the phenomenon that the expression of Artemis (Serine516) in mid-anagen and mature anagen was stronger than that in telogen and catagen, we suggest it may take roles in induced growth of mouse hair.     

Telogen skin contains an inhibitor of hair growth

We have investigated whether C57B1-6 mouse skin with all its follicles in the telogen stage of the hair cycle contains a hair-growth inhibitory activity, as opposed to skin with anagen follicles. Crude aqueous extracts of whole telogen mouse skin (TE), anagen skin (AE) or vehicle alone (V) were injected intraperitoneally into mice in which anagen had previously been induced by plucking of telogen hair follicles. Injection of TE, but not AE or V, significantly retarded the development of anagen follicles, as measured by macroscopic and quantitative microscopic hair growth parameters (skin pigmentation and thickness, appearance of trichohyaline granules) and the incorporation of tritiated thymidine into mouse skin from animals previously treated with either TE or V (skin organ culture). This inhibitory activity seemed to be localized to the epidermis and was also present in rat epidermis. We suggest that this apparently non-species-specific inhibitor present in telogen skin may play a role in regulating the hair cycle in rodents.     

Leptin of dermal adipose tissue is differentially expressed during the hair cycle and contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair

Adipose tissue encircles the lower portion of anagen hair follicles and may regulate hair cycle progression. As leptin is a major adipokine, its level of expression from the dermal white adipose tissue during hair cycle progression was studied. The result shows that leptin level is differentially expressed during hair cycle, the lowest in early anagen phase, upregulated in late anagen phase and the highest in the telogen phase. On the other hand, leptin receptor is detected in keratin 15‐positive hair bulge epithelium of both anagen‐ and telogen‐phase hair follicles of mice pelage and vibrissa hair, and hair from human scalp. Leptin contributes to adipocyte‐mediated growth inhibition of anagen‐phase vibrissa hair as demonstrated in organ culture and coculture system. Our data suggest that leptin of dermal white adipose tissue might regulate hair growth and, therefore, hair cycle progression via leptin receptor on the hair follicle epithelium.     

Ber-EP4 antigen is a marker for a cell population related to the secondary hair germ

Ber-EP4 is an antibody to a cell membrane glycoprotein of unknown function. In the skin, Ber-EP4 immunoreactivity has been reported to be localized in structures composed of basaloid epithelial cells, i.e. fetal epithelial germ cells, basal cell carcinoma, and trichoepithelioma as well as eccrine or apocrine ducts. In this study, we further characterized the follicular expression of Ber-EP4 immunoreactivity at different stages of the hair cycle of human terminal hair follicles. In addition, to clarify the location of Ber-EP4(+) cells, we compared the Ber-EP4 immunoreactivity with the expression of keratin 15 and keratin 19. Positive staining by Ber-EP4 was found in the lower part of the epithelial strand of late catagen hair follicles, in the secondary hair germ of telogen hair follicles, and in the matrix of early anagen hair follicles but not in any parts of mature anagen hair follicles. In contrast, the follicular expression of keratin 15 detected by using LHK15 antibody was restricted to two distinct parts of anagen hair follicles, i.e. the outer root sheath above the hair bulb and that of the isthmus including the bulge area, and to the outer root sheath of late catagen and telogen hair follicles. The follicular expressions of keratin 19 and that of keratin 15 were apparently superimposed, whereas keratin 15 expression was more extended. The immunoreactivity of LHK15 antibody and antikeratin 19 antibody against the secondary hair germ of telogen follicles was negative or dim. Our results suggest that Ber-EP4 reacts with the secondary hair germ and possibly a cell population related to the secondary hair germ but not with the presumptive stem cell population as revealed immunohistochemically either by the keratin 15 or keratin 19 expression.     

Procyanidin B-2, extracted from apples, promotes hair growth: a laboratory study

Summary Background We have previously reported that several selective protein kinase C (PKC) inhibitors, including procyanidin B‐2, promote hair epithelial cell growth and stimulate anagen induction. Objectives We discuss the hypothesis that the hair‐growing activity of procyanidin B‐2 is related to its downregulation or inhibition of translocation of PKC isozymes in hair epithelial cells. Methods We examined the effect of procyanidin B‐2 on the expression of PKC isozymes in cultured murine hair epithelial cells as well as PKC isozyme localization in murine dorsal skin at different stages in the hair cycle. Results We observed that procyanidin B‐2 reduces the expression of PKC‐α, ‐βΙ, ‐βΙΙ and ‐η in cultured murine hair epithelial cells and also inhibits the translocation of these isozymes to the particulate fraction of hair epithelial cells. Our immunohistochemical analyses demonstrated that PKC‐α, ‐βΙ, ‐βΙΙ and ‐η are specifically expressed in the outer root sheaths of both anagen and telogen hair follicles. The hair matrix at the anagen stage showed no positive staining for these PKC isozymes. Moderate to intense staining for PKC‐βΙ and ‐βΙΙ in the epidermis and hair follicles was observed in a telogen‐specific manner; however, expression of PKC‐α and ‐η during the telogen stage was not conspicuous. Gö 6976, an inhibitor of calcium‐dependent (conventional) PKC, proved to promote hair epithelial cell growth. Conclusions These results suggest that PKC isozymes, especially PKC‐βΙ and ‐βΙΙ, play an important role in hair cycle progression and that the hair‐growing mechanisms of procyanidin B‐2 are at least partially related to its downregulation of PKC isozymes or its inhibition of translocation of PKC isozymes to the particulate fraction of hair epithelial cells.     

Cell kinetic study of human and mouse hair tissues using anti-bromodeoxyuridine monoclonal antibody

To determine sites of cell proliferation in hair tissues, in vitro and in vivo labeling with bromodeoxyuridine (BrdU) and immunohistochemical demonstration of BrdU incorporation sites by anti-BrdU monoclonal antibody were performed on human and mouse hairs and hair follicles. The germinative area of the hair bulb of human anagen hair was divided into three portions: (A) the upper and inner portion, (B) the middle portion and (C) the lowest outer portion. A-cells intermingled with melanocytes, were regarded as germinative cells of the hair cortex. B-cells appeared to develop into Huxley's layer, cuticle of inner root sheath (IRS), and hair cuticle. C-cells seemed to develop into bulbar outer root sheath (ORS), the innermost cell (IMC) layer of the ORS and Henle's layer. The suprabulbar portion, where the ORS abruptly increased in thickness, was found to be the fourth main germinative portion (D). The ORS cells, except for the IMCs, seemed to originate mostly from the D-cells. In the late anagen phase, first, C-cells became BrdU negative, then, A- and B-cells gradually turned negative, and finally, D-cells lost their germinative activity. In catagen and telogen hair tissues, BrdU-positive cells were found in the two outer cell layers in the ORS. The structure of anagen hair tissues seems to be maintained by the coordinated mitotic activities of characteristically distributed germinative cells of various hair cell layers. The sequential cessation of mitotic activity of these cells is associated with the morphological changes from anagen through catagen to telogen. These findings were common to both human and mouse hair tissues.     

Dermal glycosaminoglycan concentration throughout hair growth cycle of rats.

The dermal glycosaminoglycan (GAG) content of rats was measured quantitatively after plucking hair from the backs of the animals, which reinitiates their hair cycle. Mast cells present around the hair follicles were counted at each stage of the hair cycle and compared with the amount of GAG in the skin. GAG reached its peak on the 6th day of the anagen stage and decreased during the catagen stage. In the telogen stage, it was about the same as it had been in the catagen stage. The same pattern was noted for the number of mast cells. The 0.5 M fraction in Dowex-1 column chromatography proved to be hyaluronic acid and its concentration throughout the hair growth cycle showed strong parallels to that of total dermal GAG.     

HMB-45抗原在毛囊黑素细胞中的表达

用单克隆抗体ＨＭＢ－４５，以ＡＰＡＡＰ法对２０份头皮活检标本的９８个毛囊组织进行染色。其中２０个毛囊为生长早期，６３个为成熟生长期，７个为退行期和８个是休止期。结果，５２个成熟生长期及２０个生长早期毛囊的黑素细胞与单克隆抗体ＨＭＢ－４５显著结合。所有退行期、休止期及工１１个成熟生长期毛囊未被染色。结果提示；ＨＭＢ－４５在毛囊中的表达随毛发生过周期而变化，且与黑素细胞功能活性状态有关。     

Plucking during telogen induces apoptosis in the lower part of hair follicles

Highly synchronized anagen development is achieved by depilation, which contrasts with spontaneous anagen development. The precise mechanism responsible for plucking-induced synchronization has not been explained. Plucking appears to mechanically injure or wound the follicle. We hypothesized that the injured hair follicle cells are removed by apoptosis, with an associated induction of synchronized anagen development. Female C57 BL/6 mice in which all the hair follicles of the back skin were in telogen were induced to enter anagen by depilation. Skin specimens from the back at 0 to 72 h after plucking were examined, and TUNEL staining and electrophoresis of isolated DNA were applied to substantiate the presence of apoptosis. Simultaneously, cell proliferation was also examined by bromodeoxyuridine (BrdU) incorporation. All methods of analysis revealed that plucking induced apoptosis of hair follicle cells in the dermal remnant, and there was a subsequent associated proliferative response that resulted in a highly synchronized anagen phase. From 12 to 36 h after plucking, hair follicle cells with weakly BrdU-positive nuclei were detected around the holes in the lower part of the follicle, which were induced by plucking. At 48 and 72 h, many cells with BrdU-positive nuclei were seen in the entire follicle, mainly in the bulb, as well as in the epidermis. Cell proliferation and apoptosis appeared to occur simultaneously after plucking. These findings mirror the events noted during the development of other organs in which the coordination of cell proliferation and apoptosis is essential for orderly restructuring events.     

Exogen hair characterization in human scalp

BACKGROUND/AIM: Classically, the hair cycle is described as a sequence of three successive phases: a hair-growth phase named anagen, a regression phase or catagen and a resting phase or telogen. In rodents, it appears that the resting hair follicle population contains also a new phase that has been identified recently as the exogen phase of the hair cycle. This phase leads to the release of the telogen club and results in hair shedding. The aim of this paper is to propose a method that is applicable to humans and that is able to discriminate the two components of the resting hair population i.e. the telogen and the exogen hair follicles. METHODS: We used non-invasive approaches to entrap exogen scalp hair into silicon-based polymers. We also extracted growing and non-growing hair with a calibrated dynamometer. We characterized differences between anagen, catagen, telogen and exogen root ends with histochemical stains and with the scanning electron microscope. Furthermore, we documented all known hair-cycle stages with the contrast-enhanced phototrichogram (CE-PTG) technique. RESULTS: We demonstrated that anagen and telogen hair are firmly anchored to the hair follicle and that cohesion forces are correlated with hair thickness. On the contrary, exogen hair are passively retained within the hair follicle. Among the resting hair population, telogen clubs retain cellular elements of the outer root sheaths that are not found on exogen hair. The specificity of the new exogen collection method was documented with the simultaneous use of the CE-PTG method: indeed anagen, catagen and telogen follicles remain unaffected by the exogen extraction procedure. CONCLUSION: Exogen hair can be sampled specifically from the human scalp with a new non-invasive method. Our data suggest that the casual levels of exogen hair, in normal individuals and under the present experimental conditions, are usually less than seven hair per cm(2).     

Alopecia areata: alterations in the hair growth cycle and correlation with the follicular pathology

A histopathological study was performed in 17 patients with alopecia areata to elucidate the changes in hair cycle dynamics. The findings confirm the view that the initial event in alopecia areata is the premature entry of anagen follicles into telogen, although some follicles survive for a time in a dystrophic anagen state. However, after re-entry into anagen takes place, growth appears to be halted in anagen III rather than anagen IV, as has previously been suggested. Follicles then return prematurely to telogen and these truncated cycles are repeated until the disease activity subsides. A new pathogenic hypothesis is presented which relates alterations in hair cycle dynamics to pathological changes within the anagen follicle and also provides an explanation for the formation of exclamation mark hairs and the non-destructive nature of the disease.     

Comprehensive analysis of FGF and FGFR expression in skin: FGF18 is highly expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice.     

The effect of topical minoxidil on hair follicular cycles of rats

To explore an easily accessible and reproducible model for examining the effect of minoxidil on hair growth, we studied the effect of minoxidil on the natural hair cycles of rats from birth to 80 days of age. During the 1st and 2nd postnatal cycles, the hair follicles grew very rapidly and the size of anagen follicles were markedly enlarged. In the 3rd cycle (50 days to approximately 100 days of age), duration of the telogen phase lasted approximately 20 days. Topical minoxidil, 1%, 3%, or 5% solution, applied on the backs of the rats from 23 days (weaning) to 80 days, induced a remarkable shortening of the telogen phase in the 3rd cycle. Although the dose-dependent response was very minimal, rats treated with 3% or 5% minoxidil showed similar effects in the 4th cycle. Minoxidil, however, did not induce prolongation of the anagen phase, but increased the rate of DNA synthesis in the anagen bulb during the 2nd and 3rd cycles. These results suggest that minoxidil specifically stimulates the secondary germ of the telogen follicles, resulting in rapid progression to anagen follicles.     

Exosomes derived from human dermal papilla cells promote hair growth in cultured human hair follicles and augment the hair‐inductive capacity of cultured dermal papilla spheres

Dermal papillae (DP) play key roles in hair growth and regeneration by regulating follicular cell activity. Owing to the established roles of exosomes (Exos) in the regulation of cell functions, we investigated whether DP‐derived Exos, especially those from three‐dimensional (3D)‐cultured DP cells, affect hair growth, cycling and regeneration. Exos derived from 3D DP (3D DP‐Exos) promoted the proliferation of DP cells and outer root sheath (ORS) cells and increased the expression of growth factors (IGF‐1, KGF and HGF) in DP cells. 3D DP‐Exo treatment also increased hair shaft elongation in cultured human hair follicles. In addition, local injections of 3D DP‐Exos induced anagen from telogen and also prolonged anagen in mice. Moreover, Exo treatment in human DP spheres augmented hair follicle neogenesis when the DP spheres were implanted with mouse epidermal cells. Similar results were obtained using Exos derived from 2D‐cultured DP cells (2D DP‐Exo). Collectively, our data strongly suggest that Exos derived from DP cells promote hair growth and hair regeneration by regulating the activity of follicular dermal and epidermal cells; accordingly, these findings have implications for the development of therapeutic strategies for hair loss.     

Hair cycle-specific expression of versican in human hair follicles

BACKGROUND: Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure. OBJECTIVES: To elucidate the implication of versican in normal human hair growth. METHODS: Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein). RESULTS: The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness. CONCLUSION: Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.     

CD10 and CD34 in fetal and adult human hair follicles: dynamic changes in their immunohistochemical expression during embryogenesis and hair cycling

Background  CD10 and CD34 have been detected in both epithelial and mesenchymal components of anagen human hair follicles.
Objectives  To analyse the expression of CD10 and CD34 in human hair follicle development as well as in different phases of the hair cycle.
Methods  Fetal and adult hair follicles at different stages of the hair cycle were examined by immunohistochemistry for CD10 and CD34.
Results  In fetal follicles, CD10 is expressed by the cells of the placodes, and CD34 by the mesenchymal cells of the dermal condensate. As the follicle matures, CD10 can be seen in the matrix cells, inner root sheath and dermal sheath. In adult follicles, the expression of CD10 in the follicular epithelium is present in anagen follicles, but tends to disappear in catagen, and is not detected in telogen. The CD10 positivity of the dermal sheath is more intense in catagen than in anagen follicles. CD34 immunostaining of the external root sheath was seen in adult anagen follicles but not in fetal follicles. This staining of the anagen outer sheath tends to disappear in catagen and is not detected in telogen.
Conclusions  CD10 and CD34 are not proteins constantly present in a specific cell type of the hair follicle, but are proteins that can be expressed by both epithelial and mesenchymal cells depending on the stage of development and hair cycle. The distribution of the immunoreactivity to CD10 in the placode and CD34 in the dermal condensate suggests a role of these proteins in initial stages of hair formation.     

The hair cycle and its regulation

The most important feature of hair growth is the cyclic activity of hair follicles in which an active phase, anagen, is succeeded by a resting phase, telogen, during which the fully formed hair remains anchored in the follicle. Between anagen and telogen there is a short transitional phase called catagen. In 1924, Trotter¹ recognized that such a cycle existed in man. Two years later, Dry² described the cycle in detail for the mouse and first used the terminology, which currently is universally accepted. A detailed account of the hair cycle in the human scalp, however, awaited the work of Kligman in 1959.³ Today, his pictures still provide classic illustrations of the sequence of events.     

Syndecan-1 is strongly expressed in the anagen hair follicle outer root sheath and in the dermal papilla but expression diminishes with involution of the hair follicle

Syndecan-1 is the prototypic member of a family of heparan sulfate-bearing cell surface proteoglycans that function in adhesion, cell-extracellular matrix interactions, migration, and proliferation. During embryogenesis, syndecan-1 expression in the epithelium is downregulated when the epithelium gives rise to motile mesenchymal cells, whereas mesenchymal syndecan-1 expression is upregulated during organ formation. In aggressive basal cell carcinomas, syndecan-1 expression is evident in the stroma. Some neoplastic cells induce stroma to meet needs for growth, and it may be the mesenchymal cells that produce and shed syndecan-1 into the stroma. The physiologic mechanism by which the hair follicle undergoes its cyclic process of involution and formation of a new active hair follicle is not well understood. Sixty scalp biopsies and a large scalp resection were evaluated for syndecan-1 expression within hair follicles in the growing (anagen), involuting (catagen), and resting (telogen) phases. Strong syndecan-1 immunoreactivity was evident in the outer root sheath (ORS) of the anagen hair follicle, but this expression diminished in intensity with the involution and resting stages in the hair follicle cycle. The diminution of syndecan-1 immunoreactivity in the ORS of involuting and resting hair follicles may be a result of terminal keratinocyte differentiation. Syndecan-1 was also present in the dermal papilla of the anagen hair follicle, where it may promote growth factor-mediated cell signaling that induces and maintains growth of the hair shaft and the inner root sheath.