The development of anin vitrohuman hair follicle organoid with a complexity similar to thatin vivo.

In vitrohair follicle (HF) models are currently limited toex vivoHF organ cultures (HFOCs) or 2D models that are of low availability and do not reproduce the architecture or behavior of the hair, leading to poor screening systems. To resolve this issue, we developed a technology for the construction of a humanin vitrohair construct based on the assemblage of different types of cells present in the hair organ. First, we demonstrated that epithelial cells, when isolatedin vitro, have similar genetic signatures regardless of their dissection site, and their trichogenic potential is dependent on the culture conditions. Then, using cell aggregation techniques, 3D spheres of dermal papilla (DP) were constructed, and subsequently, epithelial cells were added, enabling the production and organization of keratins in hair, similar to what is seenin vivo. These reconstructed tissues resulted in the following hair compartments: K71 (inner root-sheath), K85 (matrix region), K75 (companion layer), and vimentin (DP). Furthermore, the new hair model was able to elongate similarly toex vivoHFOC, resulting in a shaft-like shape several hundred micrometers in length. As expected, when the model was exposed to hair growth enhancers, such as ginseng extract, or inhibitors, such as TGF-B-1, significant effects similar to thosein vivowere observed. Moreover, when transplanted into skin biopsies, the new constructs showed signs of integration and hair bud generation. Owing to its simplicity and scalability, this model fully enables high throughput screening of molecules, which allows understanding of the mechanism by which new actives treat hair loss, finding optimal concentrations, and determining the synergy and antagonism among different raw materials. Therefore, this model could be a starting point for applying regenerative medicine approaches to treat hair loss.

Mannosylerythritol lipids ameliorate ultraviolet A-induced aquaporin-3 downregulation by suppressing c-Jun N-terminal kinase phosphorylation in cultured human keratinocytes.

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of PPAR-γ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of PPAR-γ. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Irritating effects of sodium lauryl sulfate on human primary keratinocytes at subtoxic levels of exposure.

Chemical agents that can potentially cause skin irritation are typically tested in animal models or in vitro assays of cell viability or cytokine expression. However, these methods do not always provide translatable results and are not sufficiently sensitive for subtoxicity detection. Here, we introduce the mechanical properties of keratinocytes as novel endpoints for the safety assessment of chemical agents at the subtoxicity level. Human primary keratinocytes were treated with various concentrations of sodium lauryl sulfate (SLS) and their biological properties (proliferation, membrane integrity, inflammatory response, and morphology) were observed. Their biomechanical and geometrical parameters (stiffness and surface roughness) were also investigated by atomic force microscopy. Keratinocyte morphophysiological changes and inflammatory responses were significant at ≥25 µM SLS. The keratinocytes became less stiff due to changes in the distribution of F-actin filaments and α-tubulin; these changes were significant even at lower doses of SLS (≤10 µM). The morphophysiological changes of keratinocytes were clearly seen at a relatively high dose of SLS, while the mechanical properties of keratinocytes responded linearly to SLS at lower doses. Therefore, changes in mechanical properties can be used as new endpoints for in vitro toxicity testing with keratinocytes.

Heat shock protein 90 inhibitor enhances apoptosis by inhibiting the AKT pathway in thermal-stimulated SK-MEL-2 human melanoma cell line.

BACKGROUND: Heat shock proteins (Hsps) are chaperone proteins, which are upregulated after various stresses. Hsp90 inhibitors have been investigated as adjuvant therapies for the treatment of melanoma. Thermal ablation could be a treatment option for surgically unresectable melanoma or congenital nevomelanocytic nevi, however, there is a limitation such as the possibility of recurrence. OBJECTIVE: We evaluated apoptosis in a melanoma cell line treated with the Hsp90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in hyperthermic conditions. METHODS: SK-MEL-2 cells were stimulated at 43 °C for 1 h and treated with 0, 0.1 and 1 µM 17-DMAG. We evaluated the cell viability using MTT and apoptosis with HSP 90 inhibitor. We studied the protein expression of AKT, phospho-AKT, ERK, phospho-ERK, MAPK, and phospho-MAPK, caspase 3,7,9, and anti-poly (ADP-ribose) polymerase. RESULTS: 17-DMAG significantly inhibited the proliferation of the SK-MEL-2 cells at 37 °C (0.1 µM: 44.47% and 1 µM: 61.23%) and 43 °C (0.1 µM: 49.21% and 1 µM: 63.60%), suggesting synergism between thermal stimulation and 17-DMAG. 17-DMAG treatment increased the frequency of apoptotic cell populations to 2.17% (0.1 µM) and 3.05% (1 µM) in 37 °C controls, and 4.40% (0.1 µM) and 4.97% (1 µM) in the group stimulated at 43 °C. AKT phosphorylation were activated by thermal stimulation and inhibited by 17-DMAG. CONCLUSION: Hsp90 inhibitor treatment may be clinically applicable to enhance the apoptosis of melanoma cells in hyperthermic condition.

Manifestation of atopic dermatitis-like skin in TNCB-induced NC/Nga mice is ameliorated by topical treatment of substance P, possibly through blockade of allergic inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. In this study, topically applied substance P (SP) significantly alleviated AD-like clinical symptoms in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced dermatitis in NC/Nga mice. This effect was nullified by pretreatment of the neurokinin-1 receptor (NK-1R) antagonist CP99994. SP treatment significantly reduced the infiltration of mast cells and CD3-positive T cells as well as inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and thymic stromal lymphopoietin (TSLP), in AD-like skin lesions and decreased the levels of IgE and thymus and activation-regulated chemokine in serum. This SP-induced alleviation of allergic inflammatory responses was also confirmed as reduced activation in the axillary lymph nodes (aLN) and spleen, suggesting the systemic effect of SP on immune responses in TNCB-induced NC/Nga mice. Furthermore, SP-mediated TSLP reduction was confirmed in human keratinocyte culture under pro-inflammatory TNF-α stimulation. Taken together, these results suggest that topically administered SP may have potential as a medication for atopic dermatitis.

Substance P restores normal skin architecture and reduces epidermal infiltration of sensory nerve fiber in TNCB-induced atopic dermatitis-like lesions in NC/Nga mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by intense pruritus and eczematous lesion. Substance P (SP) is an 11-amino-acid endogenous neuropeptide that belongs to the tachykinin family and several reports recently have supported the anti-inflammatory and tissue repairing roles of SP. OBJECTIVE: In this study, we investigated whether SP can improve AD symptoms, especially the impaired skin barrier function, in 2, 4, 6-trinitrochlorobenzene (TNCB)-induced chronic dermatitis of NC/Nga mice or not. METHOD: AD-like dermatitis was induced in NC/Nga mice by repeated sensitization with TNCB for 5 weeks. The experimental group designations and topical treatments were as follows: vehicle group (AD-VE); SP group (AD-SP); and SP with NK1R antagonist CP99994 (AD-SP-A) group. Histological analysis was performed to evaluate epidermal differentiation, dermal integrity, and epidermal nerve innervation in AD-like lesions. The skin barrier functions and pruritus of NC/Nga mice were evaluated by measuring transepidermal water loss (TEWL) and scratching behavior, respectively. RESULT: Topical SP treatment resulted in significant down-regulation of Ki67 and the abnormal-type keratins (K) K6, K16, and K17, restoration of filaggrin and claudin-1, marked reduction of TEWL, and restoration of basement membrane and dermal collagen deposition, even under continuous sensitization of low dose TNCB. In addition, SP significantly reduced innervation of itch-evoking nerve fibers, gelatinase activity and nerve growth factor (NGF) expression in the epidermis but upregulated semaphorin-3A (Sema3A) expression in the epidermis, along with reduced scratching behavior in TNCB-treated NC/Nga mice. All of these effects were completely reversed by co-treatment with the NK1R antagonist CP99994. In cultured human keratinocytes, SP treatment reduced expression of TGF-α, but upregulated TGF-ß and Sema3A. CONCLUSION: Topically administered SP can restore normal skin barrier function, reduce epidermal infiltration of itch-evoking nerve fibers in the AD-like skin lesions, and alleviate scratching behavior. Thus, SP may be proposed as a potential medication for chronic dermatitis and AD.

Changes in the biomechanical properties of a single cell induced by nonthermal atmospheric pressure micro-dielectric barrier discharge plasma.

Mechanical properties of a single cell are closely related to the fate and functions of the cell. Changes in mechanical properties may cause diseases or cell apoptosis. Selective cytotoxic effects of nonthermal atmospheric pressure micro-dielectric barrier discharge (DBD) plasma have been demonstrated on cancer cells. In this work, changes in the mechanical properties of a single cell induced by nonthermal atmospheric pressure micro-DBD plasma were investigated using atomic force microscopy (AFM). Two cervical cancer cell lines (HeLa and SiHa) and normal human fibroblast cells (HFBs) were exposed to micro-DBD plasma for various exposure times. The elasticity of a single cell was determined by force-distance curve measurement using AFM. Young's modulus was decreased by plasma treatment for all cells. The Young's modulus of plasma-treated HeLa cells was decreased by 75% compared to nontreated HeLa cells. In SiHa cells and HFBs, elasticity was decreased slightly. Chemical changes induced by the plasma treatment, which were observed by Raman spectroscopy, were also significant in HeLa cells compared to SiHa cells and HFBs. These results suggested that the molecular changes induced by micro-DBD plasma were related to cell mechanical changes.

Substance P induces M2-type macrophages after spinal cord injury.

The potential benefits or the tissue-damaging effects of inflammatory response after central nervous system injuries have long been disputed. Recent studies have noted that substance P (SP), a neuropeptide, plays an important role in the wound-healing process by recruiting bone marrow stem cells to the injured tissue. In this study, we examined whether SP can enhance recovery from spinal cord injury (SCI) in Sprague-Dawley rats through its known function of stem cell mobilization and/or through the modulation of inflammation. We examined proinflammatory and anti-inflammatory cytokines and markers for macrophage subtypes. SP treatment modulated the SCI microenvironment toward a more anti-inflammatory and reparative one by inducing interleukin-10 and M2 macrophages and suppressing inducible nitric oxide synthase and tumor necrosis factor-α. This modulation was achieved at 1 day much earlier than SP-stimulated bone marrow stem cells' mobilization. Early intervention of the devastating inflammatory response by SP treatment caused the lesion cavity to become filled with robust axonal outgrowth that overlaid the M2 macrophages at 2 weeks--all of which culminated in tissue sparing and improvement in functional recovery from the SCI. SP is therefore a potential anti-inflammatory modulator for the treatment of injury-induced inflammatory central nervous system disorders.