Ginsenosides in Panax ginseng Extract Promote Anagen Transition by Suppressing BMP4 Expression and Promote Human Hair Growth by Stimulating Follicle-Cell Proliferation.

Studies showing that Panax ginseng promotes hair growth have largely been conducted using mice; there are few reports on how P. ginseng affects human hair growth. In particular, little is known about its effect on the telogen to anagen transition. To determine the effect of P. ginseng on human hair growth and the transition from the telogen to the anagen phase. The effects of P. ginseng extract (PGE) and the three major ginsenoside components, Rb1, Rg1, and Re, on the proliferation of human dermal papilla cells (DPCs) and human outer root sheath cells (ORSCs) were investigated. The effects of these compounds on the cell expression of bone morphogenetic protein 4 (BMP4), fibroblast growth factor 18 (FGF18) and Noggin were assessed by real-time PCR. The effect of PGE on hair-shaft elongation was determined in a human hair follicle organ-culture system. PGE and the three ginsenosides stimulated the proliferation of DPCs and ORSCs and suppressed BMP4 expression in DPCs but did not affect FGF18 expression in ORSCs and Noggin expression in DPCs. PGE stimulated hair-shaft growth. PGE and the ginsenosides Rb1, Rg1, and Re stimulate the transition from the telogen phase to anagen phase of the hair cycle by suppressing BMP4 expression in DPCs. These compounds might be useful for promoting the growth of human hair.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Changes in the Expression of Smooth Muscle Cell-Related Genes in Human Dermal Sheath Cup Cells Associated with the Treatment Outcome of Autologous Cell-Based Therapy for Male and Female Pattern Hair Loss.

In a clinical study of autologous cell-based therapy using dermal sheath cup (DSC) cells, the treatment of hair loss showed improvements. However, the outcomes were variable. Here, correlations between marker gene expression in DSC cells and treatment outcomes were assessed to predict therapeutic efficacy. Overall, 32 DSC cell lines were used to evaluate correlations between marker gene expression and treatment outcomes. Correlations between vascular pericyte and preadipocyte marker expression and treatment outcomes were inconsistent. As smooth muscle cell markers, MYOCD correlated negatively with treatment outcomes and SRF consistently demonstrated an inverse correlation. Additionally, CALD1 correlated negatively and ACTA2 correlated inversely with treatment outcomes. DSC cell lines were divided into good and moderate/poor responders to further investigate the correlations. SRF and CALD1 were lower in a good responder compared with a moderate responder. Next, DSC cells were differentiated toward dermal papilla cells. Dermal papilla markers SOX2 and LEF1 before differentiation had moderate positive and inverse correlations with the treatment outcome, respectively. SOX2 after differentiation more consistently demonstrated a positive correlation. Significant downregulation of smooth muscle-related genes was also observed after differentiation. These findings revealed putative markers for preclinical evaluation of DSC cells to improve hair loss.

Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation.

Epidermal keratinocyte (KC) differentiation, which involves the process from proliferation to cell death for shedding the outermost layer of skin, is crucial for the barrier function of skin. Therefore, in dermatology, it is important to elucidate the epidermal KC differentiation process to evaluate the symptom level of diseases and skin conditions. Previous dermatological studies used staining or labelling techniques for this purpose, but they have technological limitations for revealing the entire process of epidermal KC differentiation, especially when applied to humans. Here, we demonstrate label-free visualization of three-dimensional (3D) intracellular morphological changes of ex vivo human epidermis during epidermal KC differentiation using stimulated Raman scattering (SRS) microscopy. Specifically, we observed changes in nuclei during the initial enucleation process in which the nucleus is digested prior to flattening. Furthermore, we found holes left behind by improperly digested nuclei in the stratum corneum, suggesting abnormal differentiation. Our findings indicate the great potential of SRS microscopy for discrimination of the degree of epidermal KC differentiation.

1-(2-Hydroxyethyl)-2-imidazolidinone, a heparanase and matrix metalloproteinase inhibitor, improves epidermal basement membrane structure and epidermal barrier function.

Daily exposure to sunlight is known to affect the structure and function of the epidermal basement membrane (BM), as well as epidermal differentiation and epidermal barrier function. The aim of this study is to clarify whether the inhibition of BM-degrading enzymes such as heparanase and matrix metalloproteinase 9 (MMP-9) can improve the epidermal barrier function of facial skin, which is exposed to the sun on a daily basis. 1-(2-hydroxyethyl)-2-imidazolidinone (HEI) was synthesized as an inhibitor of both heparanase and MMP-9. HEI inhibited not only the BM damage at the DEJ but also epidermal proliferation, differentiation, water contents and transepidermal water loss abnormalities resulting from ultraviolet B (UVB). This was determined in this study by the use of UVB-induced human cultured skins as compared with the control without HEI. Moreover, topical application of HEI improved epidermal barrier function by increasing water content and decreasing transepidermal water loss in daily sun-exposed facial skin as compared with non-treated skins. These results suggest that the inhibition of both heparanase and MMP-9 is an effective way to care for regularly sun-exposed facial skin by protecting the BM from damage.

Marked Changes in Lamellar Granule and Trans-Golgi Network Structure Occur during Epidermal Keratinocyte Differentiation.

Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation.

The topical penta-peptide Gly-Pro-Ile-Gly-Ser increases the proportion of thick hair in Japanese men with androgenetic alopecia.

BACKGROUND: A penta-peptide, Gly-Pro-Ile-Gly-Ser (GPIGS), promotes proliferation of mouse hair keratinocytes and accelerates hair growth in mice. AIM OF THIS STUDY: This study focused on the ability of the peptide to promote human hair growth. METHODS: We used a human hair keratinocyte proliferation assay and organ cultures of human hair follicle as in vitro systems. The lotions with and without the penta-peptide were administered to 22 Japanese men with androgenetic alopecia (AGA) for 4 months in a double-blind and randomized clinical study. RESULTS: The penta-peptide significantly stimulated the proliferation of human hair keratinocytes at a concentration of 2.3 µm (P < 0.01), and 5.0 µm of this peptide had a marked effect on hair shaft elongation in the organ culture (P < 0.05). The change in the proportion of thick hair (≥60 µm) compared to baseline in patients that received the peptide was significantly higher than in the placebo (P = 0.006). The change in the proportion of vellus hair (<40 µm) was also significantly lower in the peptide group than in the placebo (P = 0.029). The penta-peptide also significantly improved the appearance of baldness (P = 0.020) when blinded reviewers graded photographs of the participants according to a standardized baldness scale. No adverse dermatological effects due to treatment were noted during this clinical study. CONCLUSIONS: This penta-peptide promotes proliferation of human hair keratinocytes and hair shaft elongation of human hair follicles, in vitro. This peptide increases thick hair ratio in vivo, and this compound is useful for the improvement of AGA.

Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia.

Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 µm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P < 0.0001). The change in vellus hair proportion (<40 µm) was significantly lower in the adenosine group than that in the placebo group (P = 0.0154). The change in hair density compared with baseline of the adenosine group was also significantly higher compared with that of the placebo group (P = 0.0470). No adverse effects due to treatment were noted during this study by dermatological evaluation. Adenosine is effective in increasing the proportion of thick hair in Caucasian men with AGA as well as in Japanese men and women.

Keratinocyte-specific mesotrypsin contributes to the desquamation process via kallikrein activation and LEKTI degradation.

Kallikrein-related peptidases (KLKs) have critical roles in corneocyte desquamation and are regulated by lymphoepithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Recently, we reported cloning of a PRSS3 gene product, keratinocyte-specific mesotrypsin, from a cDNA library. We hypothesized that mesotrypsin is involved in the desquamation process, and the aim of the present study was to test this idea by examining the effects of mesotrypsin on representative desquamation-related enzymes pro-KLK5 and pro-KLK7. Incubation of mesotrypsin and these zymogens resulted in generation of the active forms. KLK activities were effectively inhibited by recombinant LEKTI domains D2, D2-5, D2-6, D2-7, D5, D6, D6-9, D7, D7-9, and D10-15, whereas mesotrypsin activity was not susceptible to these domains, and in fact degraded them. Immunoelectron microscopy demonstrated that mesotrypsin was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Proximity ligation assay showed close association between mesotrypsin and KLKs in the granular to cornified layers. Age-dependency analysis revealed that mesotrypsin was markedly downregulated in corneocyte extract from donors in their sixties, compared with younger donors. Collectively, our findings suggest that mesotrypsin contributes to the desquamation process by activating KLKs and degrading the intrinsic KLKs' inhibitor LEKTI.

Perivascular localization of dermal stem cells in human scalp.

In mammalian skin, the existence of stem cells in the dermis is still poorly understood. Previous studies have indicated that mesenchymal stem cells (MSCs) are situated as pericytes in various mammalian tissues. We speculated that the human adult dermis also contains MSC-like cells positive for CD34 at perivascular sites similar to adipose tissue. At first, stromal cells from adult scalp skin tissues showed colony-forming ability and differentiated into mesenchymal lineages (osteogenic, chondrogenic and adipogenic). Three-dimensional analysis of scalp skin with a confocal microscope clearly demonstrated that perivascular cells were positive for not only NG2, but also CD34, immunoreactivity. Perivascular CD34-positive cells were abundant around follicular portions. Furthermore, CD34-positive cell fractions collected with magnetic cell sorting were capable of differentiating into mesenchymal lineages. This study suggests that dermal perivascular sites act as a niche of MSCs in human scalp skin, which are easily accessible and useful in regenerative medicine.

Promotion of lymphatic integrity by angiopoietin-1/Tie2 signaling during inflammation.

The cutaneous lymphatic system plays a major role in tissue fluid homeostasis and inflammation of the skin. Although several lymphangiogenic factors are known to be involved in the formation of lymphatic vessels, the molecular mechanisms that maintain lymphatic integrity and control the functional drainage of interstitial fluid and resolution of inflammation remain unknown. Here we show that angiopoietin-1 (Ang1) enhances lymphatic integrity and function during inflammation. Ang1 transgenic mice under the control of keratin-14 (K14-Ang1) showed attenuated edema formation and inflammation after UV B (UVB) exposure. After UVB irradiation, blood vascular permeability was inhibited in K14-Ang1 mice compared with wild-type (WT) mice. Moreover, lymphatic vessels of WT mice were markedly enlarged and leaky in inflamed skin, whereas K14-Ang1 mice showed relatively contracted lymphatic vessels together with enhanced lymphatic vascularization. Expression of endothelial-specific tight junction molecules claudin-5 and zonula occludens protein 1 (ZO-1) was strongly down-regulated in the inflamed lymphatic vessels of UVB-exposed WT mice, whereas down-regulation of both claudin-5 and ZO-1 was blocked in UVB-exposed K14-Ang1 mice. In vitro studies revealed that the stability of lymphatic endothelial cells was enhanced in the presence of Ang1, presumably via up-regulation of claudin-5, as well as ZO-1. Claudin-5 knockdown markedly increased the permeability of lymphatic endothelial cells. Overall, our data strongly support the idea that Ang1/Tie2 signaling promotes lymphatic integrity by modulating tight junction molecule expression during inflammation.

Coenzyme Q10 protects against oxidative stress-induced cell death and enhances the synthesis of basement membrane components in dermal and epidermal cells.

Coenzyme Q10 (CoQ10), which has both energizing and anti-oxidative effects, is also reported to have antiaging action, e.g., reducing the area of facial wrinkles. However, the mechanism of its anti-aging activity is not fully established. Here, we examined the effect of CoQ10 on human dermal and epidermal cells. CoQ10 promoted proliferation of fibroblasts but not keratinocytes. It also accelerated production of basement membrane components, i.e., laminin 332 and type IV and VII collagens, in keratinocytes and fibroblasts, respectively; however, it had no effect on type I collagen production in fibroblasts. CoQ10 also showed protective effects against cell death induced by several reactive oxygen species in keratinocytes, but only when its cellular absorption was enhanced by pretreatment of the cells with highly CoQ10-loaded serum. These results suggest that protection of epidermis against oxidative stress and enhancement of production of epidermal basement membrane components may be involved in the antiaging properties of CoQ10 in skin.

RNA cleavage linked with ribosomal action.

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.

CoQ10 supplementation elevates the epidermal CoQ10 level in adult hairless mice.

We have already shown that prolonged supplementation of CoQ(10) in humans reduces the wrinkle area rate and wrinkle volume per unit area in the corner of the eye. CoQ(10) supplementation is known to increase the CoQ(10) level in serum and in many organs; however, the level of CoQ(10) in skin has not yet been fully investigated yet. We examined whether CoQ(10) intake elevates the CoQ(10) and CoQ(9) levels in epidermis, dermis, serum and other organs (kidney, heart, brain, muscle and crystalline lens) in 43-week-old hairless male mice. We also established a method using a high performance liquid chromatograph equipped with an electrochemical detector (HPLC-ECD) to simultaneously quantify CoQ(9) and CoQ(10) in the tissues. CoQ(10) (0, 1, 100 mg/kg p.o.) was administered daily for 2 weeks. CoQ(10) supplementation of 100 mg/kg increased the serum and epidermal CoQ(10) levels significantly, but did not increase the CoQ(10) levels in either dermis or other organs. In conclusion, we showed that CoQ(10) intake elevates the epidermal CoQ(10) level, which may be a prerequisite to the reduction of wrinkles and other benefits related to the potent antioxidant and energizing effects of CoQ(10) in skin.