Hajimu Oura

The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells

The effect of cultured normal human dermal papilla cells (DPCs) and conditioned medium prepared with cultured DPCs on chemotactic migration of human hair outer root sheath cells (ORSCs) was examined quantitatively. ORSCs showed significantly increased migration toward both cultured DPCs and the conditioned medium suggesting that DPCs produce and secrete a paracrine factor(s), which attracts hair follicle epithelial cells. Some soluble factors, which are reportedly produced by DPCs, such as insulin-like growth factor-I (IGF-I), hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1), were also examined. ORSCs showed dramatically increased migration toward IGF-I and HGF at concentrations of 1-10 ng/ml. On the other hand, neither VEGF nor TGF-beta1 showed any effect on the chemotaxis of ORSCs. It is interesting that all factors involving mitogenic activity did not always have chemotactic activity for ORSCs. This is the first report to establish that IGF-I and HGF have not only a growth stimulatory but also a chemotactic effect on ORSCs. In addition, the method presented here may help to simplify chemotaxis assays of any type of epithelial keratinocytes with poor mobility.

Targeted deletion of the murine corneodesmosin gene delineates its essential role in skin and hair physiology

Controlled proteolytic degradation of specialized junctional structures, corneodesmosomes, by epidermal proteases is an essential process for physiological desquamation of the skin. Corneodesmosin (CDSN) is an extracellular component of corneodesmosomes and, although considerable debate still exists, genetic studies have suggested that the CDSN gene in the major psoriasis-susceptibility locus (PSORS1) may be responsible for susceptibility to psoriasis, a human skin disorder characterized by excessive growth and aberrant differentiation of keratinocytes. CDSN is also expressed in the inner root sheath of hair follicles, and a heterozygous nonsense mutation of the CDSN gene in humans is associated with scalp-specific hair loss of poorly defined etiology. Here, we have investigated the pathogenetic roles of CDSN loss of function in the development of skin diseases by generating a mouse strain with targeted deletion of the Cdsn gene. Cdsn-deficient mouse skin showed detachment of the stratum corneum from the underlying granular layer and/or detachment within the upper granular layers due to the disrupted integrity of the corneodesmosomes. When grafted onto immunodeficient mice, Cdsn-deficient skin showed rapid hair loss together with epidermal abnormalities resembling psoriasis. These results underscore the essential roles of CDSN in hair physiology and suggest functional relevance of CDSN gene polymorphisms to psoriasis susceptibility.

Immunohistological analysis of P53 expression in human skin tumors

The p53 expression in various skin tumors was immunohistologically evaluated using two mouse monoclonal anti-p53 antibodies, PAb421 and PAb1801. The p53 expression was not detected in the normal epidermal cells. Nuclear staining suggested that the p53 expression was observed in 10 of 26 squamous cell carcinomas (SCCs) from 24 patients, in one undifferentiated carcinoma, one proliferating trichilemmal cyst, one malignant proliferating trichilemmal tumor and in one metastatic carcinoma of breast cancer. None off four cases of Bowens disease (SCC in situ) showed nuclear staining. In the SCCs, five of 20 primary lesions, three of four recurrent lesions and both of two metastatic lesions had positive nuclei. There was one case of SCC in which a primary lesion was negative but a recurrent lesion was positive. Thus, p53 expression was more frequently observed in SCCs at more clinically advanced stages. This may suggest that p53 has some relevance to progression of SCC. Nuclear staining was not detected in any of the following cases: two cases of seborrheic keratosis, one eccrine poroma, one keratoacanthoma, 11 basal cell epitheliomas, two mammary Pagets disease, three genital Pagets disease, one sebaceous carcinoma, four malignant melanomas, six lymphomas, two leukemia cutis and two angiosarcomas.

Microcystic adnexal carcinoma. Case report with an immunohistochemical study.

We report the case of 71-year-old Japanese woman with microcystic adnexal carcinoma (MAC) of the nose. Our histological and immunohistochemical observations suggest that MAC has both pilar and eccrine sweat gland differentiation.

Adenosine increases anagen hair growth and thick hairs in Japanese women with female pattern hair loss: A pilot, double-blind, randomized, placebo-controlled trial

Adenosine upregulates the expression of vascular endothelial growth factor and fibroblast growth factor‐7 in cultured dermal papilla cells. It has been shown that, in Japanese men, adenosine improves androgenetic alopecia due to the thickening of thin hair due to hair follicle miniaturization. To investigate the efficacy and safety of adenosine treatment to improve hair loss in women, 30 Japanese women with female pattern hair loss were recruited for this double‐blind, randomized, placebo‐controlled study. Volunteers used either 0.75% adenosine lotion or a placebo lotion topically twice daily for 12 months. Efficacy was evaluated by dermatologists and by investigators and in phototrichograms. As a result, adenosine was significantly superior to the placebo according to assessments by dermatologists and investigators and by self‐assessments. Adenosine significantly increased the anagen hair growth rate and the thick hair rate. No side‐effects were encountered during the trial. Adenosine improved hair loss in Japanese women by stimulating hair growth and by thickening hair shafts. Adenosine is useful for treating female pattern hair loss in women as well as androgenetic alopecia in men.

Ephrin-A3 not only increases the density of hair follicles but also accelerates anagen development in neonatal mice

BACKGROUND Ephrins are cell-membrane-bound ligands for Eph receptor tyrosine kinases (Eph). Although ephrins are known to regulate a variety of developmental processes, little is known of their role in hair development. Previously, we studied the gene expression of dermal papilla cells from androgenetic alopecia and found that ephrin-A3 was significantly down-regulated. OBJECTIVE To characterize the expression of ephrin-A3 in the hair cycle and evaluate the effect of ephrin-A3 on hair growth. METHODS We investigated gene expression and protein expression of each ephrin-As and EphAs in the skin of neonatal mice through the first and second hair cycle using quantitative PCR and immunohistochemical analysis, respectively. We also injected ephrin-A3 protein into the skin of neonatal mice and demonstrated the effect of ephrin-A3 on hair follicle development. RESULTS Expression of ephrin-A3 revealed a rapid increase at the beginning of the anagen phase, a peak during the mid-anagen, and a rapid fading during the telogen phase. In addition, we found ephrin-A3 protein was expressed in the developing hair follicles with a characteristic spatiotemporal localization. Furthermore, injection of ephrin-A3 into the skin of neonatal mice markedly accelerated the differentiation process of hair follicles. In addition, injection of ephrin-A3 unexpectedly increased the number of hair follicles. CONCLUSION These findings demonstrated that ephrin-A3 not only accelerates anagen development but also increases the density of hair follicles, and also suggested that an ephrin-A-EphA signal pathway is closely involved in hair follicle development.

IMMUNOHISTOCHEMICAL DETECTION OF P53 TUMOR SUPPRESSOR PROTEIN IN POROKERATOSIS

We examined 9 Japanese cases of porokeratosis (4 of the plaque type, 2 of disseminated superficial actinic porokeratosis, 2 of disseminated superficial porokeratosis, and one of giant porokeratosis) for the expression of p53 tumor suppressor protein immunohistochemically, using two anti‐p53 antibodies, CM1 and DO1. The same results were obtained with both antibodies. The epidermis central to the cornoid lamellae was positive in 8 of 9 specimens. On the other hand, the peripheral epidermis was positive in 2 of the 9 cases. The epidermis beneath the cornoid lamellae was positive in 3 of the 9 cases. The frequency of p53 positivity was significantly higher in the epidermis central to cornoid lamellae over that beneath or peripheral to them (Fishers exact probability test, p<0.05). The majority of squamous cell carcinoma cells arising on giant porokeratosis stained with CM1 and DO1. These data may suggest that the abnormal p53 expression has some relevance to the skin carcinogenesis of porokeratosis.

Dispersed Cell Culture of Human Sweat Duct Cells under Serum-free Conditions

Human eccrine gland duct cells were successfully cultured using a serum‐free medium, K‐GM medium. Eccrine sweat ducts were isolated from dispase treated skin specimens from palms or soles. After treatment of the isolated ducts with trypsin and EDTA, dispersed cells were cultured in K‐GM medium. In primary cultures, small colonies were seen 3 to 4 days after inoculation. Then the cells rapidly proliferated and formed large colonies with a paving stone‐like cell arrangement. During the culture, small dome shaped areas were sometimes formed in the centers of colonies. Cultures multiplied for a maximum of 7 passages. The plating efficiencies of the 1st to 6th passage cells were about 20% to 30%. Immunocytochemically, cultured cells were positively stained with anti‐carcinoembryonic antigens, K8.37 and K8.13, but not with anti‐S100 protein, anti‐HLA‐DR, 34βB4, or PKK3. An electron micrograph of the cultured cells showed a multilayer of flattened cells linked by desmosomes. These results indicate that the cultured cells possessed the staining properties compatible with those of the ductal portion of eccrine sweat glands. No contamination by other mesenchymal cells, such as fibroblasts, was seen during the culture.

Blood Vessels and Immunoreactive Substance P‐Containing Nerve Fibers in Rat Skin Treated Topically with Clobetasol Propionate, a Corticosteroid

After applying topically a cream (0.1 ml) containing corticosteroid (clobetasol propionate), on rat back skin, we examined the morphological alterations of blood vessels, substance P‐containing nerve fibers, and cutaneous mast cells. After 3, 6, 10, 15, 30, and 60 min and 4 h, the skin treated was cut out with a sharp knife after killing the animals. The skin pieces were processed into conventional histological sections cut vertically and examined by staining immunohistochemically with anti‐substance P serum, by staining with toluidine blue for mast cell granules, and by estimating morphometrically the average areas of vascular cavity and the number of substance P fibers in the dermis. In the dermis and subcutaneous tissue of untreated skin, we found many immunoreactive SP‐containing nerve fibers and mast cells in close association. Three to ten min after the treatment, the average area of the vascular cavities steadily increased, and SP‐positive fibers became less frequent in the dermis. In concomitant with those events, cutaneous mast cells discharged their granules. Thereafter, the average area of vascular cavities gradually decreased to a minimum at 4 h after the treatment. In contrast, both SP‐containing fibers and mast cells reestablished their initial states after the same duration.

Novel ALK5 inhibitor TP0427736 reduces TGF-β induced growth inhibition in human outer root sheath cells and elongates anagen phase in mouse hair follicles

BACKGROUND Androgenic alopecia (AGA) occurs as a result of the contraction of the anagen phase because of the action of androgens on hair follicles. TGF-β production from dermal papillae is enhanced by androgens, and growth inhibition of hair-follicle cells is induced by TGF-β, and the hair cycle progresses from the anagen phase to the catagen phase. We investigated both the in vitro and in vivo potency of the newly identified ALK5 inhibitor TP0427736 {6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole}. METHODS For in vitro study, kinase inhibitory activity was evaluated with ELISA, and inhibitory activity against TGF-β-induced Smad2/3 phosphorylation in A549 cells and TGF-β-induced growth inhibition of human outer root sheath cells were assayed using ELISA. For in vivo study, we used a mouse model that had been synchronized through dorsal hair depilation. RESULTS TP0427736 inhibited ALK5 kinase activity with an IC50 of 2.72nM; this effect was 300-fold higher than the inhibitory effect on ALK3. In cell-based assays, TP0427736 inhibited Smad2/3 phosphorylation in A549 cells and decreased the growth inhibition of human outer root sheath cells. The topical application of TP0427736 significantly decreased Smad2 phosphorylation in mouse skin, and its repeated application suppressed the shortening of average hair follicle length during the transition from the late anagen phase to the catagen phase. CONCLUSIONS TP0427736, a potent ALK5 inhibitor with appropriate in vitro and in vivo profiles, may serve as a potential new therapy for AGA.　.BACKGROUND Androgenic alopecia (AGA) occurs as a result of the contraction of the anagen phase because of the action of androgens on hair follicles. TGF-β production from dermal papillae is enhanced by androgens, and growth inhibition of hair-follicle cells is induced by TGF-β, and the hair cycle progresses from the anagen phase to the catagen phase. We investigated both the in vitro and in vivo potency of the newly identified ALK5 inhibitor TP0427736 {6-[4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-5-yl]-1,3-benzothiazole}. METHODS For in vitro study, kinase inhibitory activity was evaluated with ELISA, and inhibitory activity against TGF-β-induced Smad2/3 phosphorylation in A549 cells and TGF-β-induced growth inhibition of human outer root sheath cells were assayed using ELISA. For in vivo study, we used a mouse model that had been synchronized through dorsal hair depilation. RESULTS TP0427736 inhibited ALK5 kinase activity with an IC50 of 2.72 nM; this effect was 300-fold higher than the inhibitory effect on ALK3. In cell-based assays, TP0427736 inhibited Smad2/3 phosphorylation in A549 cells and decreased the growth inhibition of human outer root sheath cells. The topical application of TP0427736 significantly decreased Smad2 phosphorylation in mouse skin, and its repeated application suppressed the shortening of average hair follicle length during the transition from the late anagen phase to the catagen phase. CONCLUSIONS TP0427736, a potent ALK5 inhibitor with appropriate in vitro and in vivo profiles, may serve as a potential new therapy for AGA.　.