Toshimasa Jindo

The Expression of Endothelin‐1 and Its Binding Sites in Mouse Skin Increased after Ultraviolet B Irradiation or Local Injection of Tumor Necrosis Factor α

Endothelin (ET)‐1 is a 21‐amino acid peptide which has vasoconstrictor and growth regulatory activity. Recently, cultured keratinocytes have been reported to express ET‐1 and its receptor when irradiated by ultraviolet (UV) B. In order to further understand the role of ET‐1 in vivo during UVB‐induced inflammation, we examined the localization, intensity and time course of the expression levels of ET‐1 and its binding sites in UVB‐exposed BALB/c mouse skin. Frozen and paraffin sections prepared from mouse skin 48 h after treatment with UVB irradiation (0.36 or 0.72 J/cm2) or after injection with tumor necrosis factor (TNF)‐α (1.0 μg) or interleukin (IL)‐1α (0.05 μg) were incubated with monoclonal anti‐ET‐1 IgG and then visualized by peroxidase staining. In normal skin, faint ET‐1 immunoreactivity was observed in the epidermis, pilosebaceous structures and blood vessels. Upon exposure to UVB irradiation or administration of TNF‐α injection or IL‐1α injection, such immunoreactivity was found to be significantly enhanced. Subsequently, the frozen sections were incubated with 125I ET‐1 for 30 min, and visualized by autoradiographic technique. In normal skin, ET‐1 weakly bound to the skin, while UVB irradiation and TNF‐α injection significantly enhanced ET‐1 binding in the epidermis, pilosebaceous structures and blood vessels. Time course experiments (1, 2, 4 and 7 days) indicated that ET‐1 immunoreactivity and ET‐1 binding peaked 1 or 2 days after UVB irradiation or TNF‐α injection. These results suggest that the up‐regulated expression of ET‐1 and its binding sites in the epidermis and pilosebaceous structures may act as an autocrine/paracrine factor during UVB‐induced inflammation.

The effect of hepatocyte growth factor/scatter factor on human hair follicle growth

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on human hair follicle growth was examined using a serum-free organ culture system. The DNA synthesis in human hair follicles and elongation of the hair shaft were measured subsequent to the follicle isolation and culture at 31 degrees C in 95% O2-5% CO2 for 72 h. Results showed that HGF/SF significantly increased 3H-thymidine (P < 0.001) incorporation and hair follicle length (P < 0.05). The effect of HGF/SF was dose-dependent with a maximal stimulation at 10 ng/ml.

Organ culture of human hair follicles in serum-free medium

Human hair follicles were cultured in serum-free media at 31‡C in an atmosphere containing 95% O2 and 5% CO2. Results showed that the length of the cultured hair increased time dependently for 96 h. Histological findings revealed that the hair germinative cells maintained their normal morphology throughout the 96 h culture period. DNA synthesis in the hair bulb also increased time dependently for 96 h. Autoradiographs of 3H-thymidinelabelled follicles indicated that they were localized in the germinative cells below Aubers critical line. The effects of minoxidil sulphate on DNA synthesis in this culture system were concentration dependent. Minoxidil sulphate at concentrations of 10−10,10−9 and 10−8M significantly increased DNA synthesis compared with DNA synthesis in the control medium. Autoradiographs of the follicles cultured in 10−10M minoxidil sulphate showed that 3H-thymidine localized primarily in the germinative cells below Aubers critical line. These results suggest that this organ culture system may be useful for studying DNA synthesis by hair germinative cells in serum-free media.

Organ culture conditions of human hair follicles

Experimental results revealed that although [3H]thymidine uptake in the hair bulb increased time dependently for 12 days under normal culture conditions (95% air-5% CO2 at 37 degrees C), striking morphological changes occurred in the hair bulb cells as demonstrated by histological findings. As such, organ culture conditions applicable to human hair follicles were studied utilizing observations from both histology and DNA synthesis. We found that culture conditions of 95% O2-5% CO2 at 31 degrees C were superior when compared to normal culture conditions for cultures of human hair follicles when attempting to maintain the normal morphology of hair germinative cells. The hair bulb and the germinative cells successfully maintained their normal morphology throughout the 96 and 48 h culture period, respectively. Autoradiographs of [3H]thymidine-labeled follicles showed localization in the germinative cells below Aubers critical line. Hair bulb DNA synthesis increased time dependently for 96 h after culture initiation. Under conditions of 95% O2-5% CO2 at 31 degrees C, the synthesis of DNA in hair germinative cells was observed. Such an organ culture method may prove useful for studies on the human hair growth mechanism.

Organ culture of mouse vibrissal hair follicles in serum-free medium.

We developed a method for organ culture of mouse vibrissal hair follicles in a serum‐free medium. Cultures conducted at 31°C in 95% O2‐5% CO2 were found to be suitable for the follicles, with several findings of considerable interest pertaining to hair growth. During the 96 h culture period, the length of the isolated follicles significantly increased; the hair bulb cells maintained their normal morphology; and DNA and protein synthesis within the bulb increased time‐dependently. Furthermore, autoradiography showed that 3H‐thymidine‐labeling was localized in the matrix cells below Aubers critical line in the hair bulb; 3H‐leucine‐labeling was found in the epithelial region; and 35S‐cysteine‐labeling was detected in the cortex of hair, particularly in the keratogenous zone. These results indicate that the culture system using mouse vibrissal hair would be potentially useful as an effective model for examination of hair growth.

The effect of various cytokines on hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional polypeptide which acts as a mitogen, motogen or morphogen depending on the biological context. In this study, we examined the effect of HGF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2-5%CO2 for 72 h in the presence of various cytokines or growth factors. DNA, protein synthesis and elongation of the hair shaft in the hair follicles were measured. Among the agents tested, only HGF significantly increased hair follicle length (P < 0.001) and 3H-thymidine (P < 0.001) incorporation. The effect of HGF was dose-dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF. These results indicate that HGF is able to promote hair growth and may have clinical utility in this regard.

Effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in hair bulb cells

We analysed the effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in human hair germinative cells cultured in serum-free media. Epidermal growth factor and gamma interferon were found to inhibit DNA synthesis slightly, while strong inhibition was demonstrated by doxorubicin, cytosine arabinoside and tetradecanoyl-phorbolacetate. Basic fibroblast growth factor had very little influence on DNA synthesis. This organ culture model in serum-free media is a useful method by which to examine the effects of various cytokines and drugs on DNA synthesis in hair germinative cells and/or to study the pathogenesis of various alopecia diseases.