High migratory activity of dermal sheath cup cells associated with the clinical efficacy of autologous cell-based therapy for pattern hair loss.

BACKGROUND: Autologous cell-based therapy using dermal sheath cup (DSC) cells was reported as a new treatment for male and female pattern hair loss. However, the mechanisms underlying its action remain unclear. OBJECTIVE: We investigated the mechanisms underlying the efficacy of DSC cells in cell-based therapy. METHODS: We conducted multivariate analysis to categorize individuals based on treatment response as responders and non-responders. The differentially expressed genes in DSC cells from the two groups were evaluated using bulk transcriptome, quantitative polymerase chain reaction, and single-cell transcriptome analyses. We performed live cell imaging combined with immunostaining to characterize the DSC subpopulation associated with responders. RESULTS: We identified nine and three genes as high efficacy (HE) and low efficacy (LE) marker genes, respectively. The HE subpopulations were enriched for cell migration-related genes in single-cell analysis. In contrast, the LE subpopulation was enriched for basement membrane and vasculature-related genes. Moreover, DSC cells in culture were immunocytochemically and morphologically heterogeneous, expressing characteristic factors. Furthermore, live cell imaging showed that DSC cells expressing integrin subunit alpha 6 (ITGA6), an HE subpopulation gene, had markedly higher mobility than those expressing the LE subpopulation genes collagen type IV or CD36. CONCLUSIONS: ITGA6-positive DSC cells, with superior migratory activity, may contribute to cell-based therapy by promoting cell migration into nearby hair follicles.

Gene Expression Profiling of the Intact Dermal Sheath Cup of Human Hair Follicles.

Cells that constitute the dermal papillae of hair follicles might be derived from the dermal sheath, the peribulbar component of which is the dermal sheath cup. The dermal sheath cup is thought to include the progenitor cells of the dermal papillae and possesses hair inductive potential; however, it has not yet been well characterized. This study investigated the gene expression profile of the intact dermal sheath cup, and identified dermal sheath cup signature genes, including extracellular matrix components and bone morphogenetic protein-binding molecules, as well as transforming frowth factor beta 1 as an upstream regulator. Among these, gremilin-2, a member of the bone morphogenetic protein antagonists, was found by in situ hybridization to be highly specific to the dermal sheath cup, implying that gremlin-2 is a key molecule contributing to maintenance of the properties of the dermal sheath cup.

Effect of oral intake of winged bean extract on a skin lichenification model: evaluation by microarray analysis.

Winged bean (WB), Psophocarpus tetragonolobus, is a tropical legume, the potential of which is not fully understood. We found that 5-week oral administration of a WB seed extract inhibited wrinkle formation induced by repeated tape stripping (TS) as a model of lichenification in human chronic eczematous dermatitis. To elucidate the mechanism of the effect of WB on this model, we applied microarray analysis. Hierarchical clustering revealed that each experimental group formed a distinct cluster, suggesting the presence of a distinct gene expression profile among the three groups of non-TS, TS, and TS with oral administration of WB extract (TS/WB). Gene ontology analysis showed that several gene groups with keratinization and mitosis were significantly upregulated by TS, while other groups with ATP synthesis and glycolysis were significantly downregulated by TS/WB. Moreover, WB extract influenced a number of genes related to epidermal differentiation and inflammation. This suggests that these changes inhibited wrinkle formation by TS.

Inflammatory mediator TAK1 regulates hair follicle morphogenesis and anagen induction shown by using keratinocyte-specific TAK1-deficient mice.

Transforming growth factor-beta-activated kinase 1 (TAK1) is a member of the NF-kappaB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial-mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7(fl/fl)K5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7(fl/fl)K5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7(fl/fl)K14-Cre-ER(T2)). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.

Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.

Restricted expression of caspase-14 in differentiating keratinocytes suggests the involvement of caspase-14 in terminal differentiation. We purified active caspase-14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764-fold with a yield of 9.1%. Purified caspase-14 revealed the highest activity on WEHD-methylcoumaryl-amide (MCA), although YVAD-MCA, another caspase-1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N-terminal and C-terminal analyses demonstrated that the large subunit consisted of Ser(6)-Asp(146) and N-terminal of small subunit was identified as Lys(153). We successfully developed an antiserum (anti-h14D146) directed against the Asp(146) cleavage site, which reacted only with active caspase-14 but not with procaspase-14. Furthermore we confirmed that anti-h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti-h14D146 staining was mostly restricted to the cornified layer and co-localized with some of the TUNEL positive-granular cells in the normal human epidermis. UV radiation study demonstrated that caspase-3 was activated and co-localized with TUNEL-positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase-14 activation in response to UV. Our study revealed tightly regulated action of caspase-14, in which only the terminal differentiation of keratinocytes controls its activation process.

Identification of novel hair-growth inducers by means of connectivity mapping.

The aim of this study was to identify novel inducers of hair growth using gene expression profiling at various stages of hair-growth induction. First, we analyzed gene expression at the onset of hair growth in mice induced by cyclosporin A (CsA), a well-known hair-growth inducer, using DNA microarray analysis. The results unveiled genes involved in the step-by-step progression of hair growth, including increases in melanin biosynthesis and decreases in immune response at d 2 and the subsequent stimulation of cell proliferation at d 4, followed by the up-regulation of hair specific keratins at d 7 after CsA treatment. With the use of the connectivity map (Cmap), agents that had a similar "gene signature" to that of the profiles of CsA-treated mice were identified. Several agents, including CsA, were identified by the Cmap and were evaluated for hair induction activity in vivo. One of the proposed agents, fluphenazine (from the d 2 signature) actually induced hair growth in vivo (ED(50): 2 mM for single application), and the subsequent application of 5 mM iloprost (from the d 4 signature) significantly enhanced the hair-growth effect of fluphenazine. From these results, Cmap analysis was proven to be a useful method that connects gene expression profiles of complicated biological processes, such as hair-growth induction, to effective agents.

Hair follicle regeneration using grafted rodent and human cells.

Hair follicle regeneration involves epithelial-mesenchymal interactions (EMIs) of follicular epithelial and dermal papilla (DP) cells. Co-grafting of those cellular components from mice allows complete hair reconstitution. However, regeneration of human hair in a similar manner has not been reported. Here, we investigated the possibility of cell-based hair generation from human cells. We found that DP-enriched cells (DPE) are more critical than epidermal cells in murine hair reconstitution on a cell number basis, and that murine DPE are also competent for hair regeneration with rat epidermal cells. Co-grafting of human keratinocytes derived from neonatal foreskins with murine DPE produced hair follicle-like structures consisting of multiple epidermal cell layers with a well-keratinized innermost region. Those structures expressed hair follicle-specific markers including hair keratin, and markers expressed during developmental stages. However, the lack of regular hair structures indicates abnormal folliculogenesis. Similar hair follicle-like structures were also generated with cultured human keratinocytes after the first passage, or with keratinocytes derived from adult foreskins, demonstrating that epidermal cells even at a mature stage can differentiate in response to inductive signals from DP cells. This study emphasizes the importance of EMI in follicular generation and the differentiation potential of epidermal keratinocytes.

Hazard identification on a single cell level using a laser beam.

This research shows a novel method for hazard identification of a chemical and UV light on a single cell level with a laser probe beam. The laser probe beam was passed through interface of cell membrane/culture medium of a cultured human hepatoblastoma cell line HepG2. Deflection of the laser probe beam, which was induced by changes in concentration gradients due to the active materials movement across the cell membrane, was monitored. When a toxic hazard existed, a living cell was expected to be killed or injured, or cellular behaviors to be changed greatly. Then, the changing deflection signal from the living cell would become unchanged or altered in a different way. This was successfully demonstrated with cytotoxicity of UV light and H(2)O(2). Most of the cultured HepG2 cells showed changing deflection signals after 10 min illumination of UV-visible light longer than 370 nm, while almost all HepG2 cells showed unchanged deflection signal after 10 min illumination of UV-visible light with wavelength longer than 330 nm. The results suggested that UV light between 330-370 nm could kill the cells. Additions of H(2)O(2) solution with different concentrations to the cell cultures caused the changing deflection signal from a living cell either unchanged or changed in different trend, suggesting toxicity of H(2)O(2) to the cells. The results from the beam deflection detection agreed well with those obtained by the conventional trypane blue method.

Expression profiling and cellular localization of genes associated with the hair cycle induced by wax depilation.

The hair cycle is a highly regulated process controlled by multiple factors. Systematic analysis of gene expression patterns in each stage of the hair cycle would provide information useful for understanding this complicated process. To identify genes associated with the hair cycle, we used DNA microarray hybridization to analyze sequential gene expression patterns in mouse skin following hair cycle synchronization by wax depilation. Messenger RNA levels in mouse skin at various times after depilation were compared with those prior to depilation (resting phase). According to their expression patterns, upregulated genes were categorized into four groups: early anagen, middle anagen, late anagen/early catagen, and middle/late catagen, and processes that take place in each stage were evaluated. We identified 12 new components that are specifically expressed in the hair follicle, 11 genes in anagen including carbonic anhydrase 6, cytokeratin 12, and matrix metalloproteinase-11 in catagen that were confirmed using in situ hybridization. The strategy used here allowed us to identify unknown genes or process previously not suspected to have a role in hair biology. These analyses will contribute to elucidating the mechanisms of hair cycle regulation and should lead to the identification of novel molecular targets for hair growth and/or depilation agents.

[Diagnosis of vascular complications at the puncture site after cardiac catheterization].

OBJECTIVES: Cardiac catheterization is increasingly used for the diagnosis or treatment of coronary artery disease. Previous studies that revealed the incidence of complications such as arteriovenous fistula and pseudoaneurysm were based on retrospective analysis of cohorts referred to vascular surgery. This study was designed to determine the incidence of arteriovenous fistula and pseudoaneurysm after percutaneous transluminal angiography. METHODS: All 557 consecutive patients undergoing cardiac catheterization were examined by ultrasonography from March 1, 2001 to April 1, 2002, to investigate the occurrence of arteriovenous fistula and pseudoaneurysm at the puncture site. RESULTS: Pseudoaneurysm was found in 16 patients (2.9%), and arteriovenous fistula in 12 patients (2.2%). Pseudoaneurysm in 7 patients (43.8%) and arteriovenous fistula in 6 patients (50.0%) were diagnosed only by ultrasonic examination. There were more female patients (9 patients, 56.3%) than male with pseudoaneurysm (p < 0.01). The puncture site was located after the division of the deep femoral artery and superficial femoral artery in all patients with complications. CONCLUSIONS: Ultrasonic examination was useful for diagnosis of complications such as arteriovenous fistula and pseudoaneurysm after cardiac catheterization.

A potential suppressor of TGF-beta delays catagen progression in hair follicles.

TGF-beta plays important roles in the induction of catagen during the hair cycle. We examined whether TGF-beta2 could activate a caspase in human hair follicles. Using active caspase-9 and -3 specific antibodies, we found that TGF-beta2 activated these caspases in two regions, the lower part of the hair bulb and the outer layer of the outer root sheath. In addition, we searched for a plant extract that can effectively suppress TGF-beta action. We found that an extract of Hydrangea macrophylla reduced synthesis of a TGDbeta-inducible protein. We confirmed that the extract has a potential to promote hair elongation in the organ culture system. Furthermore, it delayed in vivo progression of catagen in a mouse model. Our results suggest that the induction of catagen by TGF-beta is mediated via activation of caspases and that a suppressor of TGF-beta could be effective in preventing male pattern baldness.

Involvement of transforming growth factor-beta2 in catagen induction during the human hair cycle.

The involvement of transforming growth factor-beta isoforms in the induction of the regressing phase (catagen) of human hair follicles were examined in vivo. In the growing phase (anagen), transforming growth factor-beta1 was detected at the hair cuticle and connective tissue sheath. Transforming growth factor-beta2 was restricted to the outermost cell layer of the outer root sheath. Transforming growth factor-beta3 was observed in the precortical hair matrix of anagen hair follicles. During the anagen-catagen transition phase, strong transforming growth factor-beta2 immunoreactivity appeared in the lower bulb matrix cells adjacent to the dermal papilla. In addition, transforming growth factor-beta2 and transforming growth factor-beta type II receptor were colocalized in the regressing epithelial strands, where terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling-positive apoptotic cells were also found. Transforming growth factor-beta1 and transforming growth factor-beta3 were mostly negative in the strand. Using an organ culture system, we investigated whether transforming growth factor-beta2 and its antagonists affected the transition process. Elongation of hair was significantly suppressed by transforming growth factor-beta2. Next, a neutralizing antibody and fetuin, a potent transforming growth factor-beta antagonist was tested. In the presence of the antibody as well as fetuin, hair follicles were markedly elongated in a concentration-dependent manner. These results strongly suggest that transforming growth factor-beta2 plays an essential part in the induction of the catagen phase of the human hair cycle.