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Metabolic studies on isolated hair follicles: hair follicles engage in aerobic glycolysis and do not demonstrate the glucose fatty acid cycle.

The matrix cells of the hair follicle have one of the highest rates of cell division in the mammalian body, but their fuel metabolism is poorly understood, due mainly to the difficulty in obtaining viable intact follicles from the skin. We have previously shown that viable and intact rat hair follicles can be isolated by shearing, and in this study we now report on their fuel metabolism. In this study we have shown that the hair follicle exhibits aerobic glycolysis, in that of the total glucose utilized by the hair follicle, only 10% is oxidized to CO2. We have also shown that, in the absence of glucose, the hair follicle is capable of utilizing other fuels such as palmitate and beta-hydroxybutyrate. However, neither palmitate or beta-hydroxybutyrate had any effect on the rate of glucose utilization or on [U-14C] glucose oxidation, showing that glucose sparing via the glucose fatty acid cycle does not operate in the hair follicle. Measurements of glucose flux through the pentose phosphate pathway accounted for only 3% of the total glucose utilized by the hair follicle, although this value represented 32% of the total glucose oxidized. Both palmitate and beta-hydroxybutyrate inhibited glucose flux through the pentose phosphate pathway.

3-Hydroxybutyric Acid↗

Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations.

BACKGROUND: CD1d belongs to a family of antigen presenting molecules that are structurally and distantly related to the classic major histocompatibility complex class I (MHC I) proteins. However, unlike MHC I molecules, which bind protein antigens, CD1d binds to lipid and glycolipid antigens. CD1d is expressed by cells of lymphoid and myeloid origin, and by cells outside of the lymphoid and myeloid lineages, such as human keratinocytes of psoriatic skin. AIMS: To investigate whether CD1d is also expressed in sun exposed skin and in the immuno-privileged anagen hair follicle. MATERIALS/METHODS: CD1d immunoreactivity was studied in human scalp skin and hair follicles of healthy women in situ by immunofluorescent and light microscopic immunohistology. Skin biopsies were obtained from normal human scalp containing mainly anagen VI hair follicles from women (age, 53-57 years) undergoing elective plastic surgery. RESULTS: CD1d showed strong immunostaining in human scalp skin epidermis, pilosebaceous units, and eccrine glands. In the epidermis, CD1d was strongly expressed by basal and granular keratinocytes. In hair follicles, CD1d was expressed in the epithelial compartment and showed hair cycle related alterations, with an increase in the anagen and a reduction in the catagen and telogen phases. CONCLUSIONS: These results suggest that CD1d plays a role in human scalp skin immunology and protection against lipid antigen rich infectious microbes. They also raise the question of whether keratinocytes of the immuno-privileged anagen hair follicle can present lipid antigens to natural killer T cells. These data could help provide new strategies for the manipulation of hair related disorders.

Antigens, CD1↗

Expression of the heat shock protein-27 in the adult human scalp skin and hair follicle: hair cycle-dependent changes.

BACKGROUND: Heat shock protein (HSP) is a molecular chaperone involved in protein folding, assembly, and transport and in the regulation of cell growth and differentiation. HSP27 protein is expressed in murine hair follicle (HF) and in human skin during fetal development. In this investigation we hypothesized that HSP27 protein is expressed in the human scalp skin and its expression in HF changes with the transitions form anagen --> catagen --> telogen stages. METHODS: To test this hypothesis, the immunoreactivity of HSP27 protein was examined in human scalp skin by immunofluorescent method. A total of 50 normal human scalp skin biopsy specimens were examined (healthy women, age 53-57 years). In each case, 50 HF were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). RESULTS: HSP27 protein expression was prominent in human scalp anagen, and weak in both catagen and telogen HFs. Within HF, HSP27 protein immunoreactivity was prominent in the outer root sheath, inner root sheath, precorteocytes, and corteocytes of the hair shaft. In addition, HSP27 protein expression was prominent in the epidermis, sebaceous glands, sweat glands, and arrector pili muscles. LIMITATIONS: Only some types of heat shock proteins are known to date. Also, our knowledge about the exact molecular mechanisms involved in the interactions among these protein and other molecular chaperones is still incomplete. CONCLUSIONS: Our investigation reports, for the first time, the expression patterns of HSP27 in human scalp skin and HF. The differential expression of HSP27 during HF cycling suggests its possible roles in human HF biology.

Adult↗

Physiology of the vellus hair follicle: hair growth and sebum excretion.

The growth of vellus hair and the secretion of sebum from vellus hair follicles were measured on the forehead, cheek, chest, shoulder and back of healthy men and women aged 15-30 years. Hair growth was assessed by computerized image-analysis of photographs and sebum excretion by the use of Sebutape followed by image analysis. The density of vellus hairs and the percentage of growing hairs were higher on the face than on the thorax (439 hairs/cm2 with 49% growing hairs on the forehead compared with 85 hairs/cm2 with 31.5% growing hairs on the back). The rate of growth ranged from 0.03 mm/day on the forehead to 0.13 mm/day on the back. The maximum length of vellus hair significantly decreased with age; otherwise hair growth was not affected by age or sex. Some variations in hair growth and sebum secretion were observed over a period of 3 months, but no consistent rhythms were detected. There was no obvious link between vellus hair growth and sebum excretion.

Adolescent↗

Ageing-associated large-scale deletions of mitochondrial DNA in human hair follicles.

Hair follicles plucked from the bi-temporal region of the scalp of 433 Chinese subjects of different ages were used for the examination of ageing-associated mutations of human mitochondrial DNA (mtDNA). By use of PCR techniques, we detected the 4,977 bp and 7,436 bp deletions of mtDNA in hair follicles from aged individuals. The frequencies of occurrence of both mtDNA deletions were found to increase with age of the subject. Moreover, we employed a semi-quantitative PCR method to determine the proportion of the 4,977 bp deleted mtDNA (dmtDNA) in hair follicles. The results showed that the average proportion of the 4,977 bp dmtDNA in hair follicles were 0.05% +/- 0.01%, 0.00%, 0.55% +/- 0.05%, 0.52% +/- 0.24%, 0.65% +/- 0.17%, 1.33 +/- 0.25%, and 1.89% +/- 0.81% for the subjects in the age groups of 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-99, respectively. Furthermore, we screened all the subjects harboring the 4,977 bp and/or 7,436 bp deletions for tandem duplications in the D-loop region of mtDNA by PCR with back-to-back primers. The results showed that none of the previously reported tandem duplications were present in all the hair follicles examined. This indicates that tandem duplications do not predispose to large-scale deletions of mtDNA. However, the data suggest that mtDNA deletions occur and accumulate in hair follicles during human ageing. As hair follicles can be easily and non-invasively obtained from the human, we suggest that the aged-dependent accumulation of dmtDNAs in hair follicles may be used for the monitoring of human ageing process.

Adolescent↗

A possible role for Langerhans cells in the removal of melanin from early catagen hair follicles.

Hair pigmentation is coupled to the hair follicle growth cycle. A common feature of catagen is the translocation of melanin from the matrix to the dermal papilla of the hair follicle. However, the mechanism whereby this pigment, not incorporated into the hair shaft, is removed from the hair bulb during early catagen is poorly understood. Routine ultrastructural examination of four normal scalp specimens revealed a rare hair follicle in early catagen. Close study of the hair bulb of this catagen follicle revealed a Langerhans cell in the process of transferring pigment from the matrix to the dermal papilla. This cell also contained numerous characteristic Langerhans granules (LG) (also known as Birbeck granules). Interestingly, these granules were intimately associated with melanosomes: so intimate, in fact, that melanosomes appeared to have been endocytosed by LG. This unique demonstration of removal of hair follicle melanin by Langerhans cells during early catagen and of pigment uptake by Langerhans cells by endocytosis into LG, suggests one way by which 'unused' pigment can be removed from the hair follicle during catagen.

Adolescent↗

Compositionally different desmosomes in the various compartments of the human hair follicle.

Hair follicles are complex organs of the skin, in morphological and ontogenic continuity with the epidermis. We have examined the location of desmosomal cadherins and desmosomal plaque proteins in the hair follicle of adult and fetal human scalp skin by immunohistochemistry and have established a localization "map" of the hair follicle. Using antibodies against the plaque proteins desmoplakin I and II, plakoglobin, and plakophilin 1, we have found that these occur in most, if not all hair follicle desmosomes, whereas plakophilin 2 was absent, except in the basal cells of the outer root sheath, where a weak reactivity was found. By contrast, the desmosomal cadherins were mostly differentially synthesized, displaying a complicated map. While desmocollin Dsc3 was detected in all cell types examined, Dsc1 was detected only in the outer root sheath companion cell layer and the inner root sheath, and Dsc2 showed practically a mutually exclusive presence. Desmoglein Dsg2 was observed in basal cells of the outer root sheath as well as in the central cell layers of the subinfundibular outer rood sheath, matrix cells and trichocytes, in partial overlap with the otherwise different immunopositive reactions of Dsg1 and Dsg3. We have also determined when these proteins are synthesized during fetal hair follicle development. The differential molecular composition of desmosomes is discussed in relation to possible functional differences between the individual cell types.

Antibody Specificity↗

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle.

Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle.

Cells, Cultured↗

Isolation of a novel population of multipotent adult stem cells from human hair follicles.

Hair follicles are known to contain a well-characterized niche for adult stem cells: the bulge, which contains epithelial and melanocytic stem cells. Using human embryonic stem cell culture conditions, we isolated a population of adult stem cells from human hair follicles that are distinctively different from known epithelial or melanocytic stem cells. These cells do not express squamous or melanocytic markers but express neural crest and neuron stem cell markers as well as the embryonic stem cell transcription factors Nanog and Oct4. These precursor cells proliferate as spheres, are capable of self-renewal, and can differentiate into multiple lineages. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. Most of the Oct4-positive cells in human skin were located in the area highlighted by cytokeratin 15 staining in vivo. Our data suggest that human embryonic stem cell medium can be used to isolate and expand human adult stem cells. Using this method, we isolated a novel population of multipotent adult stem cells from human hair follicles, and these cells appear to be located in the bulge area. Human hair follicles may provide an accessible, autologous source of adult stem cells for therapeutic application.

Cell Culture Techniques↗

Role of hair papilla cells on induction and regeneration processes of hair follicles.

Hair dermal papilla cells are specialized mesenchymal cells that exist in the dermal papilla located at the bottom of hair follicles. These cells play pivotal roles in hair formation, growth, and cycling. Hair follicle formation is usually directed by an aggregation of dermal mesenchymal cells, the origin of dermal papilla cells, in the embryonic skin. We noticed that cultured dermal papilla cells also have hair-forming activity and do not lose the activity even after long-term cultivation, if they are cultured with conditioned medium from keratinocytes obtained from the sole or with a medium containing fibroblast growth factor. The secreted factors from keratinocytes and fibroblast growth factor are, therefore, important for maintaining the cellular properties of dermal papilla cells. Even if the hair bulb, including the hair matrix and the dermal papilla, has been removed from vibrissal follicles in vivo, the new hair matrix and papilla can regenerate from the rest of the follicle, and eventually a hair shaft regrows. It has been reported that hair bulb regeneration does not occur when the lower half of a hair follicle is removed. However, new hair bulbs were formed in the remaining upper halves of vibrissal follicles if the amputated follicles had been implanted under the kidney capsule. The formed bulbs were small and pelage-type, not large vibrissa-type. Histological studies showed that the new dermal papillae were derived from dermal sheath cells surrounding upper follicular epidermis, and the new hair matrices were produced from the follicular epidermis. Moreover, the upper halves of vibrissal follicles reformed large vibrissa-type bulbs when they were associated with dermal papillae or cultured papilla cells and implanted in the kidney. Thus, dermal papilla cells and probably dermal sheath cells have the ability to induce and form hair bulbs under preferred environmental conditions. Attempts to identify the genes and proteins associated with hair-forming activity of dermal papilla cells have been carried out. We and other groups successfully isolated the molecules that were specifically expressed in dermal papilla cells. The nature of the hair-producing factors could be understood through the studies of these molecules.

Animals↗

Lectin histochemistry of glycoconjugates in the feline hair follicle and hair.

The distribution of glycoconjugate in the feline hair follicle and hair was studied by light and electron microscopic histochemical methods. The hair apparatus was found to contain considerable amounts of complex carbohydrates with different saccharide residues (alpha-D-mannose, beta-D-glucose, alpha-L-fucose, beta-N-acetyl-D-glucosamine). Variations of those were detected in the plasma membrane of the hair follicle cells during the course of their differentiation and keratinization, namely, alph-D-glucose, alpha-L-fucose and beta-N-acetyl-D-glucosamine in the suprabulbar and bulbar regions. The reaction level of sialic acid residues in the plasma membrane decreased in some cell layers during the course of differentiation. The results obtained from the present study indicated that interaction between saccharide residues of neutral carbohydrates and sialyl groups during the anagen phase might contribute to cell keratinization in hair follicles and hairs. It is discussed whether the existence of glycogen in outer root sheath cells might enable these cells to provide other hair apparatus cells with energy when necessary. Moreover, it became obvious from variations in sialyl residue distribution that cell differentiation processes terminate first of all in Huxley's and Henle's layers within the suprabulbar region of the hair follicle, as followed by the hair cortex.

Animals↗

Reconstitution of hair follicle development in vivo: determination of follicle formation, hair growth, and hair quality by dermal cells.

Combinations of cultured and uncultured epidermal and dermal cell preparations from newborn and perinatal mice were grafted onto the backs of athymic nude mouse hosts to elucidate the cellular requirements for skin appendage formation. All epidermal populations studied, including a total epidermal keratinocyte preparation from trypsin-split skin, developing hair follicle buds isolated from epidermis, and preformed hair follicles isolated from dermis, make haired skin when grafted with fresh dermal cells. Only pre-formed hair follicles produce haired skin on grafts without an additional dermal component. Hair follicle buds grafted alone or with cultured dermal cells will reconstitute skin but without appendage formation. Thus, cells or factors present in fresh, but not cultured, dermal cells are essential for supporting hair growth from budding follicles, whereas more developed (pre-formed) follicles appear to contain all the necessary components for hair formation. Dissociation of isolated hair follicles by trypsin/ethylenediaminetetraacetic acid prior to grafting is permissive for hair growth, suggesting that follicle cells can be re-induced or reassociate in vivo. Dermal papilla cells, microdissected from rat vibrissal follicles and cultured for up to 14 passages, stimulate hair growth from follicle buds and influence the quality of hair growth from pre-formed hair follicles. Thus, dermal papilla cells maintain inductive capacity in culture and contribute to the reconstituted skin. This reconstitution model should be useful for identifying cell populations within the hair follicle compartment necessary for hair growth and for examining the effects of specific gene products on hair follicle growth and development in vivo.

3T3 Cells↗

Dynamic ultrastructural changes of the connective tissue sheath of human hair follicles during hair cycle.

Ultrastructural changes of the connective tissue sheath (CTS), including the hyaline membrane, of human hair follicles during the hair cycle, were studied in normal scalp skin specimens. In early anagen, the CTS was composed of a thin basal lamina and surrounding collagen tissue. The collagen tissue gradually thickened during the development of the hair and hair follicle. In mature anagen hair follicles, the collagen tissue was separated into three layers. The inner collagen layer, just outside the basal lamina, was thin and composed of collagen fibres running longitudinally parallel to the hair axis. The middle collagen layer was very thick with its collagen fibres running transversely against the hair axis and surrounding the inner hair tissue. Many fibroblasts were present among the collagen fibres in the middle layer, whereas the inner layer contained almost none. In the outer collagen layer, collagen fibres ran in various directions parallel to the outer surface of the outer root sheath cells. In late anagen, the basal lamina became very thick. In catagen, the basal lamine and the inner collagen layer became corrugated and showed oedematous change and degeneration. Surrounding fibroblasts showed active production of new collagen fibres, which seemed to fill the spaces left by the retraction of the hair follicle and hyaline membrane. These ultrastructural changes of the CTS show that there may be dynamic metabolic changes of the connective tissue around human hair follicles during the hair cycle.

Adolescent↗

[Effects of traditional Chinese herbs on growth of mouse hair follicles and hair bulb cells in vitro].

OBJECTIVE: To investigate the effect of water soluble extracts of traditional Chinese herbs on growth of mouse hair follicles and hair bulb cells in vitro. METHODS: Mouse hair follicles and hair bulb cells were cultured in Williams E medium with (experimental groups) or without (control group) water soluble extracts of Chinese herbs; the experimental group was further divided into mixture and single herb groups. Hair growth was observed by microscopy and growth activity of hair bulb cells was detected by MTT colorimetric assay. RESULT: On day 7 of culture, the hair growth in the mixture groups was faster than that in the control group (P<0.05). On day 3 and 5 of culture, the cell growth activity in the mixture groups was greater than that in the control group (P<0.05). While the hair growth and the cell growth activity between the single herb groups and the control group were not significantly different. CONCLUSION: The water soluble extracts of mixed traditional Chinese medicines can promote the growth of mouse hair in vitro and stimulate the proliferation of hair bulb cells; while those of the single traditional Chinese herb have no effect.

Angelica sinensis↗

Evidence that activation of protein kinase A inhibits human hair follicle growth and hair fibre production in organ culture and DNA synthesis in human and mouse hair follicle organ culture.

We have investigated the possibility that protein kinase A (PKA) may play a part in regulating the activity of human and mouse hair follicles in whole organ culture. Human hair follicles were isolated from facial skin by microdissection, and hair follicle and hair fibre length measurements were made daily during suspension culture. Incubation of human hair follicles with dibutyryl-cAMP (db-cAMP) resulted in a dose-dependent inhibition of total cumulative follicle growth (IC50 = 100 mumol/L, 85% inhibition at 1 mmol/L). db-cAMP (0.5 mmol/L) also caused a rapid, partial inhibition of follicular DNA synthesis (20.3% inhibition at 6 h, 48.0% inhibition at 24 h). Human hair follicle growth was inhibited by the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and Ro 20-1724, and by the adenylate cyclase activator, forskolin. In addition, db-cAMP inhibited DNA synthesis in organ cultures of whisker follicles isolated from neonatal mice by microdissection. Taken together, these findings indicate that agents which increase cAMP levels are potent inhibitors of human and mouse hair follicle growth, and suggest that PKA may play a part in the regulation of hair follicle activity in vivo.

1-Methyl-3-isobutylxanthine↗

A method for measuring the various constituents of the human hair follicle.

Hair follicles from scalp biopsies (temporal and parietal regions) were isolated by microdissection. This technique allows preservation of the whole structure of the follicle in its fibrous sheath, or isolation of certain elements: bulb and dermal papilla. Each follicle is examined by transmission optical microscopy and its image is digitized into sixty-four grey levels by an image analyser. Follicle images are memorized on a hard disc, then recalled individually for measurement. The image analysis consists of thresholding, interactive selecting, then measurement of the following elements: diameter of the hair follicle, volume of the bulb, height of the keratogenous zone, mean diameter of the hair and size of the dermal papilla. These parameters were related to a clinical classification (terminal, dystrophic, vellus). This morphometric study constitutes an objective approach which is different from, but complementary, to the classic trichogram (telogen/anagen).

Adult↗

Human hair follicles and cultured hair follicle keratinocytes as indicators for individual differences in carcinogen metabolism.

Benzo(a)pyrene (BP)-metabolism in freshly isolated human hair follicles, cultured hair follicle keratinocytes and cells cultured from human bronchial epithelium was analysed by high performance liquid chromatography. All three types of tissues resulted in quantitatively comparable amounts of the most important organic solvent-soluble metabolites: 9,10-dihydrodiol-BP, 7,8-dihydrodiol-BP, quinones, and phenols. Besides these metabolites two early eluting compounds were detected: one possibly is BP-3-yl-hydrogen sulfate, the other probably consists of one or more tetrols. Water-soluble metabolites were quantitatively unimportant in both types of cultured cells and appeared to be primarily glucuronide and sulfate conjugates with the monohydroxides and the 7,8-dihydrodiol of BP. This metabolic pattern is compared to that of monocytes and lymphocytes which have been used frequently in population studies and with data from other types of human epithelial cells. It is concluded that human hair follicles and cultured keratinocytes from these organs are useful for detection of individual differences in carcinogen metabolism.

Benzo(a)pyrene↗

Keratin expression provides novel insight into the morphogenesis and function of the companion layer in hair follicles.

Hair follicles cycle between stages of growth (anagen) and metabolic quiescence (telogen) throughout life. In mature follicles, transition from telogen back into anagen involves the activation, proliferation, and differentiation of epithelial stem cells located in the bulge, a specialization of the outer root sheath. Recent studies identified keratin 6a (K6a) transcripts as enriched in bulge epithelial stem cells in mouse skin. We used messenger RNA probes, antibodies, a LacZ reporter mouse model, and whole-mount staining assays to investigate the regulation of mK6a during mouse postnatal hair cycling, and compare it to mK75, a companion layer (Cl) marker. We find that mK75 regulation parallels that of inner root sheath (IRS) markers, with expression onset at anagen IIIa above the new hair bulb and subsequent spreading towards the bulge. Although also occurring in the Cl, mK6a expression begins at anagen IIIb in differentiating cells located proximal to the bulge, and subsequently spreads towards the hair bulb. mK6a and mK75 thus exhibit temporally distinct, and spatially opposed, expression patterns in the Cl during postnatal anagen. These findings provide novel insight into the morphogenesis and properties of the Cl, and raise the distinct possibility that it is an integral part of the IRS compartment.

Animals↗