Expression of decorin throughout the murine hair follicle cycle: hair cycle dependence and anagen phase prolongation

Decorin is a prototypical member of the small leucine‐rich proteoglycan (SLRP) family, which is involved in numerous biological processes. The role of decorin, as a representative SLRP, in hair follicle morphogenesis has not been elucidated. We present our initial findings on decorin expression patterns during induced murine hair follicle (HF) cycles. It was found that decorin expression is exclusively restricted to the epidermis, outer root sheath and sebaceous glands during the anagen phase, which correlates with the upregulation of decorin mRNA and protein expression in depilated murine dorsal skin. Furthermore, we used a functional approach to investigate the effects of recombinant human decorin (rhDecorin) via cutaneous injection into HFs at various murine hair cycle stages. The local injection of rhDecorin (100 μg/ml) into the hypodermis of depilated C57BL/6 mice at anagen delayed catagen progression. In contrast, rhDecorin injection during the telogen phase caused the premature onset of anagen, as demonstrated by the assessment of the following parameters: (i) hair shaft length, (ii) follicular bulbar diameter, (iii) hair follicle cycling score and (iv) follicular phase percentage. Taken together, our results suggest that decorin may modulate follicular cycling and morphogenesis. In addition, this study also provides insight into the molecular control mechanisms governing hair follicular epithelial–mesenchymal interactions.     

Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells

Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell‐based co‐cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF‐1, two well‐known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.     

Desmocollin 1 expression and desmosomal remodeling during terminal differentiation of human anagen hair follicle: an electron microscopic study

The terminal differentiation (TD) program of keratinocytes of the human hair follicle (HF) occurs with specific temporal and spatial features in the various layers of the inner root sheath (IRS) and in the innermost layer of the outer root sheath (companion layer). This process is characterized by complex nuclear and cytoplasmic morphological changes, accompanied by profound modifications in intercellular junctions. As no correlation exists between the structure and the molecular composition of desmosomes during TD of the IRS/companion unit, the aim of our study was to investigate by transmission electron microscopy the remodeling of desmosomes in keratinizing cells of these compartments. By immunogold post embedding technique, we studied in anagen HFs the modulation of the synthesis of desmocollin 1 (Dsc1), a transmembrane glycoprotein specifically synthesized in the IRS and in the companion layer. Dsc1 immunoreactivity was actually confined to these compartments and tended to increase just before the level of TD, particularly in the Henle's layer and in the IRS cuticle. In Huxley's layer, the immunolabeling was patchy and in the companion layer Dsc1 synthesis was detected above the level of keratinization of Huxley's layer. In the whole IRS, concomitantly with TD, there was an abrupt and almost complete disappearance of Dsc1 synthesis. An asymmetric distribution of Dsc1 was noticed (i) between cells at different stages of differentiation and (ii) between cells belonging to layers with different spatial/temporal features of TD. Our results show that the ultrastructural modifications of desmosomes during TD of HF are paralleled by the modulation of the synthesis of desmocollin 1.     

Comparison between the expression of basement membrane zone antigens of human interfollicular epidermis and anagen hair follicle using indirect immunofluorescence

BACKGROUND: The composition of the basement membrane zone (BMZ) or dermal-epidermal junction in the interfollicular skin has been well documented. However, little is known about the BMZ or connective tissue-epithelial junction along the hair follicle. OBJECTIVES: To determine whether the BMZ antigens in the interfollicular epidermis are also present in the BMZ of the anagen hair follicle and to compare whether the expression and distribution of the BMZ components vary between the interfollicular epidermis and the anagen follicle and within different regions of the hair follicle. METHODS: Longitudinal cryostat sections of scalp margin specimens from four adult patients undergoing cosmetic surgery, and without known pathology were stained with a panel of monoclonal and polyclonal antibodies to different BMZ constituents using standard indirect immunofluorescence. RESULTS: All the BMZ antigens found in the normal interfollicular epidermis were expressed in the anagen follicle; however, there were regional variations in the intensity and patterns of fluorescence. All the antigens were expressed in a continuous linear pattern along the BMZ of the interfollicular skin, the infundibulum, and the middle part of the hair follicle. Differences were observed in the lower follicle and the hair bulb. There was continuous expression throughout the BMZ of the follicle of laminin-1 and collagen IV, but in contrast, expression of other antigens decreased down the lower follicle. There was weak or even negative staining with antibodies to alpha 6 beta 4 integrin, laminin-5, anchoring filaments, and type VII collagen in the outer aspect of the bulb compared with the hair papilla. In addition, there were special patterns observed along the bilateral middle and lower follicle. CONCLUSIONS: Despite the common embryological origin between the interfollicular epidermis and the hair follicle, there is variation in the expression of the BMZ antigens. This may be explained by the histological specialization and functional requirements that reflect the dynamic hair growth cycle.     

Effect of Plantago asiatica L. extract on the anagen phase in human hair follicle dermal papilla cells

Background

The hair growth cycle consists of the anagen, catagen, and telogen phases, and hair follicle dermal papilla (HDP) cells of human hair play a role in the initiation and maintenance of the anagen phase. Reduction in HDP cells contributes to hair loss; however, the limited treatment options are associated with negative side effects. Therefore, a naturally derived substance with hair loss-preventing properties is needed.

Aim

We investigated the hair growth-stimulating activities of Plantago asiatica L. extract (PAE) and its molecular mechanism in HDP cells.

Methods

Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution. Relative mRNA and protein expression levels of hair growth factors were determined using quantitative real-time polymerase chain reaction and western blotting, respectively. Additionally, a tube formation assay was performed in human umbilical vein endothelial cells (HUVEC).

Results

Plantago asiatica L. extract significantly increased the cell proliferation and expression of hair growth factors, including keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2) and MYC, in HDP cells. Moreover, PAE led to the accumulation of β-catenin by promoting the phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) at Ser9 and cAMP response element-binding protein (CREB) at Ser133 via phosphorylation of extracellular signal-regulated kinase (ERK) (Thr202/Tyr204). PAE also increased tube formation in HUVECs, which promoted angiogenesis for the anagen phase.

Conclusions

Plantago asiatica L. extract amplified tube formation and production of growth factors (KGF, VEGF) via the activation of GSK-3β/β-catenin and mitogen-activated protein kinase (MAPK)/CREB signaling pathways, demonstrating its potential to safely promote hair growth by inducing the anagen phase.     

Transforming growth factor-beta receptor II is preferentially expressed in the companion layer of the human anagen hair follicle

BACKGROUND: Transforming growth factor (TGF)-beta is a multifunctional growth factor with multiple roles in skin including hair follicle development and cycling, where it regulates cell proliferation, differentiation and apoptosis, as well as in wound healing. While TGF-beta receptor I (TGF-beta RI) and receptor II (TGF-beta RII) expression helps define early human hair follicle morphogenesis, expression in the adult human hair follicle remains to be established. OBJECTIVES: To assess TGF-beta receptor expression in human scalp anagen hair follicles. METHODS: Immunohistochemical and double immunofluorescence analysis of TGF-beta RI and RII was conducted on frozen sections of haired human scalp obtained from 10 healthy individuals. RESULTS: TGF-beta RI expression was detected in the outer root sheath of anagen hair follicles while TGF-beta RII was expressed almost exclusively in the companion layer of inner root sheath and less so in premedulla keratinocytes. Both receptors were colocalized in the companion layer of the proximal and mid follicle. CONCLUSIONS: The well-described role of TGF-beta in keratinocyte apoptosis during catagen is likely to involve anagen-specific hair follicle components including the companion layer, as this layer provides the slippage plane supporting the inner root sheath and hair shaft as they ascend to the skin surface. Results of this study suggest that the colocalization of TGF-beta RI/RII complexes at the companion layer would facilitate TGF-beta signalling at this site to regulate apoptosis of the companion layer keratinocytes, facilitating shrinkage/contraction of this cell layer during hair follicle regression/catagen.     

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.     

Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer

Minoxidil induces new hair growth in approximately one-third of patients with androgenetic alopecia after 1 year of treatment. With several conflicting reports in the literature based on small-scale studies, the current study aimed to clarify whether organ culture of human scalp anagen VI hair follicles is a suitable in vitro test system for reproducing, and experimentally dissecting, the recognized in vivo hair-growth-promoting capacity of minoxidil. Hair shaft elongation was studied in terminal anagen VI hair follicles microdissected from the occipital scalp of 36 healthy adults. A total of 2300 hair follicles, approximately 65 per individual, were tested using modifications of a basic organ culture protocol. It is shown here that minoxidil does not significantly increase hair shaft elongation or the duration of anagen VI in ex vivo culture despite several enhancements on the conventional methodology. This disparity to what is seen clinically in minoxidil responders may be explained by the following: (i) use of occipital (rather than frontotemporal or vertex) hair follicles; (ii) use of, already maximally growing, anagen VI hair follicles; (iii) a predominance of hair follicles from minoxidil unresponsive-donors; (iv) use of minoxidil rather than its sulfate metabolite; and/or (v) use of a suboptimal minoxidil dosage. This disparity questions the usefulness of standard human hair follicle organ culture in minoxidil research. Unexpectedly, minoxidil even inhibited hair shaft elongation in the absence of insulin, which may indicate that the actual hair-growth-modulatory effects of minoxidil depend on the concomitant local presence/absence of other growth modulators.     

Cholesterol homeostasis: Links to hair follicle biology and hair disorders

Lipids and lipid metabolism are critical factors in hair follicle (HF) biology, and cholesterol has long been suspected of influencing hair growth. Altered cholesterol homeostasis is involved in the pathogenesis of primary cicatricial alopecia, mutations in a cholesterol transporter are associated with congenital hypertrichosis, and dyslipidaemia has been linked to androgenic alopecia. The underlying molecular mechanisms by which cholesterol influences pathways involved in proliferation and differentiation within HF cell populations remain largely unknown. As such, expanding our knowledge of the role for cholesterol in regulating these processes is likely to provide new leads in the development of treatments for disorders of hair growth and cycling. This review describes the current state of knowledge with respect to cholesterol homeostasis in the HF along with known and putative links to hair pathologies.     

Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cells

Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.     

Expression of nerve growth factor mRNA and its translation products in the anagen hair follicle

The cellular localization of NGF mRNA and its translation products have been identified in ovine hair follicles. NGF mRNA was detected in the proliferating cells of the follicle bulb and differentiating cells of the suprabulbar region, but was absent from the outer root sheath. Western analysis revealed the presence of a 73 kDa NGF prohormone in extracts of ovine flank skin, but the mature 13 kDa NGF was absent. Immunohistochemical analysis with antibodies specific to mouse NGF and a pro-NGF specific domain localized the NGF prohormone to outer root sheath cells in the upper bulb region of the follicle, adjacent to the zone of keratinization. Antibody binding was also associated with the luminal epithelium of the apocrine sweat gland and the pilary canal of the follicle at its junction with the epidermis. These observations, together with the reported presence of high- and low-affinity NGF receptors in the follicle, implicate the NGF prohormone-responsive neuronal system in the regulation of hair growth.     

Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles

Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast-like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment. In an attempt to simplify hair follicle papilla cell culture methodology for new laboratory personnel, we have made significant refinements to the current method. Our method requires only two simple manipulations to isolate hair follicle papilla from intact isolated hair follicles. This very rapid and easy method isolates clean and intact hair follicle papillae. Together with their attachment via scratching to the growth surface, the isolation and cultivation of this important hair follicle component can now be achieved easily by the laboratory newcomer. The method relies for its simplicity on the removal of the hair follicle papilla from the outside of the intact hair follicle rather than via internal manipulations from within the hair follicle.     

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.     

The innermost cell layer of the outer root sheath in anagen hair follicle: Light and electron microscopic study

Summary To investigate cell differentiation in the outer root sheath (ORS) of the human anagen hair follicle, scalp skin specimens from individuals with normal hair were examined using light and electron microscopes. In the bulbar portion, the ORS was composed of two cell layers. The cells in the outer layer gradually increased in number upwards and finally underwent so-called trichilemmal keratinization, which proceeded toward the hair canal. On the other hand, the inner cells in the bulb formed a single cell layer along the outside of Henle's layer during cell differentiation; this unique layer was referred to as the innermost cell (IMC) layer of the ORS. With the use of hematoxylin and eosin stain, at the suprabulbar portion, where Henle's cells were keratinizing, an eosinophilic substance was deposited in the inner (Henle's) side of the IMC cytoplasm. The IMCs gradually became entirely eosinophilic and often produced keratohyaline granules. Ultrastructurally, the IMCs of the ORS showed an oblong shape forming a regularly arranged single-cell layer along the keratinizing Henle's layer and accumulated tonofilaments in the cytoplasm. They produced a few small electron-dense keratohyaline granules and were keratinized at the level at which Henle's layer still preserved its cell structure. From these findings, it is suggested that there are two types of keratinization of the ORS: trichilemmal keratinization and IMC keratinization.     

Immunohistochemical study of angiotensin receptors in human anagen hair follicles and basal cell carcinoma

BACKGROUND: Angiotensin receptors are the specific receptors of angiotensin II of the renin-angiotensin system. However, expression of the receptors in hair follicles has not been determined. OBJECTS: To clarify the expression and localization of angiotensin receptors in human anagen hair follicles and basal cell carcinomas. METHODS: We studied immunohistochemically the expression of angiotensin type 1(AT1) and type 2 (AT2) receptors in human anagen hair follicles and in 16 cases of basal cell carcinoma (BCC) (nine of solid BCC of the circumscribed type, two of adenoid BCC, five of BCC with follicular differentiation). RESULTS: Our experiments demonstrated the localization of AT1 in the inner root sheath and the inner layers of the outer root sheath. In BCC, positive staining with AT1 was revealed in the tumour cells of basal cell carcinoma with follicular differentiation. CONCLUSIONS: AT1 may have a role in association with follicular keratinization. Studying AT1 distribution may be useful in understanding the pathophysiology of human hair follicles and the hair follicle-associated tumours.     

A newly discovered linkage between proteoglycans and hair biology: decorin acts as an anagen inducer

Proteoglycans have been suggested to play pivotal roles in hair biology. Decorin is a prototypical member of the small leucine‐rich proteoglycan family, which is involved in numerous biological processes. However, the role of decorin in the hair cycle has not been elucidated. Moreover, the effects of decorin on the activities of many growth factors are complex, and it is hard to predict whether decorin would affect hair growth or the hair cycle positively or negatively. Jing et al. focused on the potential role of decorin in the hair cycle and found that decorin is highly expressed in the epidermis, in hair follicle epithelial cells and in dermal papilla cells in the anagen phase. The expression of decorin was decreased during catagen to telogen, except for the bulge region. Exogenous administration of decorin accelerated anagen and delayed catagen transition as a positive regulator of the hair cycle. Because TGF‐β is one of the androgen‐induced pathogenic factors in androgenetic alopecia, this study provides clues to understand the pathogenesis and new therapeutic targets of hair loss.