Injectable platelet-rich fibrin promotes proliferation and trichogenic inductivity of dermal papilla cells through activating TGF-ß/Smad signaling pathway.

Dermal papilla cell (DPC) belongs to a specialized mesenchymal stem cell for hair follicle regeneration. Maintaining the ability of DPCs to stimulate hair in vitro culture is important for hair follicle morphogenesis and regeneration. As the third generation of platelet concentrate, injectable platelet-rich fibrin (i-PRF) is a novel biomaterial containing many growth factors and showing promising effects on tissue reconstruction. We aimed to explore the influences of i-PRF on the proliferative, migratory, as well as trichogenic ability of DPCs and compared the effects of i-PRF and platelet-rich plasma (PRP), the first generation of platelet concentrate. Both PRP and i-PRF facilitated DPCs proliferation, and migration, along with trichogenic inductivity as well as stimulated the TGF-ß/Smad pathway, while the impacts of i-PRF were more significant than PRP. A small molecule inhibitor of TGF-beta receptor I, Galunisertib, was also applied to treat DPCs, and it rescued the impacts of i-PRF on the proliferative, migratory, trichogenic inductivity, and proteins-associated with TGF-ß/Smad pathway in DPCs. These findings revealed that i-PRF had better effects than PRP in enhancing the proliferative, migratory, and hair-inducing abilities of DPCs by the TGF-ß/Smad pathway, which indicated the beneficial role of i-PRF in hair follicle regeneration.

Role of Dermal Factors Involved in Regulating the Melanin and Melanogenesis of Mammalian Melanocytes in Normal and Abnormal Skin.

Mammalian melanin is produced in melanocytes and accumulated in melanosomes. Melanogenesis is supported by many factors derived from the surrounding tissue environment, such as the epidermis, dermis, and subcutaneous tissue, in addition to numerous melanogenesis-related genes. The roles of these genes have been fully investigated and the molecular analysis has been performed. Moreover, the role of paracrine factors derived from epidermis has also been studied. However, the role of dermis has not been fully studied. Thus, in this review, dermis-derived factors including soluble and insoluble components were overviewed and discussed in normal and abnormal circumstances. Dermal factors play an important role in the regulation of melanogenesis in the normal and abnormal mammalian skin.

Presence of immunoglobulin E-expressing antibody-secreting cells in the dermis close to bullous pemphigoid lesions.

Antibody-secreting cells (ASCs) produce immunoglobulin (Ig) G and IgE autoantibodies in secondary lymphoid organs. Evidence also suggests their existence in the skin in various chronic inflammatory conditions, and in association with CXCL12 and CXCL13, they regulate the recruitment/survival of ASCs and germinal center formation to generate ASCs, respectively. However, the presence of IgG and IgE in bullous pemphigoid (BP) lesions needs to be addressed. Here, we aimed to analyse BP skin for the presence of IgG and IgE and the factors contributing to their generation, recruitment, and persistence. Skin samples from 30 patients with BP were stained to identify ASCs and the immunoglobulin type they expressed. The presence of tertiary lymphoid organ (TLO) elements, which generate ASCs in non-lymphoid tissues, and the chemokines CXCL12 and CXCL13, which regulate the migration/persistence of ASCs in lymphoid tissues and formation of TLOs, respectively, were evaluated in BP skin. BP skin harboured ASCs expressing the two types of antibodies IgG and IgE. ASCs were found in high-grade cellular aggregates containing TLO elements: T cells, B cells, CXCL12+ cells, CXCL13+ cells and high endothelial venules. IgG+ ASCs were detected among these aggregates, whereas IgE+ ASCs were dispersed throughout the dermis. CXCL12+ fibroblast-like cells were located close to ASCs. The inflammatory microenvironment of BP lesions may contribute to the antibody load characteristic of the skin of patients with BP by providing a site for the presence of ASCs. CXCL13 and CXCL12 expression may contribute to the generation and recruitment/survival of ASCs, respectively.

Phloroglucinol Enhances Anagen Signaling and Alleviates H2O2-Induced Oxidative Stress in Human Dermal Papilla Cells.

Phloroglucinol (PG) is one of the abundant isomeric benzenetriols in brown algae. Due to its polyphenolic structure, PG exhibits various biological activities. However, the impact of PG on anagen signaling and oxidative stress in human dermal papilla cells (HDPCs) is unknown. In this study, we investigated the therapeutic potential of PG for improving hair loss. A non-cytotoxic concentration of PG increased anagen-inductive genes and transcriptional activities of ß-Catenin. Since several anagen-inductive genes are regulated by ß-Catenin, further experiments were performed to elucidate the molecular mechanism by which PG upregulates anagen signaling. Various biochemical analyses revealed that PG upregulated ß-Catenin signaling without affecting the expression of Wnt. In particular, PG elevated the phosphorylation of protein kinase B (AKT), leading to an increase in the inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK3ß) at serine 9. Treatment with the selective phosphoinositide 3-kinase/AKT inhibitor, LY294002, restored the increased AKT/GSK3ß/ß-Catenin signaling and anagen-inductive proteins induced by PG. Moreover, conditioned medium from PG-treated HDPCs promoted the proliferation and migration of human epidermal keratinocytes via the AKT signaling pathway. Subsequently, we assessed the antioxidant activities of PG. PG ameliorated the elevated oxidative stress markers and improved the decreased anagen signaling in hydrogen peroxide (H2O2)-induced HDPCs. The senescence-associated ß-galactosidase staining assay also demonstrated that the antioxidant abilities of PG effectively mitigated H2O2-induced senescence. Overall, these results indicate that PG potentially enhances anagen signaling and improves oxidative stress-induced cellular damage in HDPCs. Therefore, PG can be employed as a novel therapeutic component to ameliorate hair loss symptoms.

Effects of pulsed electrical stimulation on α-smooth muscle actin and type I collagen expression in human dermal fibroblasts.

Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.

Epidermal ET-1 signal induces activation of resting hair follicles by upregulating the PI3K/AKT pathway in the dermis.

The prevalence of alopecia has increased recently. Hair loss is often accompanied by the resting phase of hair follicles (HFs). Dermal papilla (DP) plays a crucial role in HF development, growth, and regeneration. Activating DP can revive resting HFs. Augmenting WNT/ß-catenin signaling stimulates HF growth. However, the factors responsible for activating resting HFs effectively are unclear. In this study, we investigated epidermal cytokines that can activate resting HFs effectively. We overexpressed ß-catenin in both in vivo and in vitro models to observe its effects on resting HFs. Then, we screened potential epidermal cytokines from GEO DATASETs and assessed their functions using mice models and skin-derived precursors (SKPs). Finally, we explored the molecular mechanism underlying the action of the identified cytokine. The results showed that activation of WNT/ß-catenin in the epidermis prompted telogen-anagen transition. Keratinocytes infected with Ctnnb1-overexpressing lentivirus enhanced SKP expansion. Subsequently, we identified endothelin 1 (ET-1) expressed higher in hair-growing epidermis and induced the proliferation of DP cells and activates telogen-phase HFs in vivo. Moreover, ET-1 promotes the proliferation and stemness of SKPs. Western blot analysis and in vivo experiments revealed that ET-1 induces the transition from telogen-to-anagen phase by upregulating the PI3K/AKT pathway. These findings highlight the potential of ET-1 as a promising cytokine for HF activation and the treatment of hair loss.

Combatting ageing in dermal papilla cells and promoting hair follicle regeneration using exosomes from human hair follicle dermal sheath cup cells.

Dermal papilla cells (DPCs) undergo premature ageing in androgenetic alopecia and senescent alopecia. As critical components of hair follicle reconstruction, DPCs are also prone to senescence in vitro, resulting in a diminished hair follicle inductivity capacity. Dermal sheath cup cells (DSCCs), a specific subset of hair follicle mesenchymal stem cells, intimately linked to the function of DPCs. The primary objective of this research is to investigate the anti-ageing effect of exosomes derived from DSCCs (ExoDSCCs ) on DPCs. Exosomes were utilized to treat H2 O2 -induced DPCs or long-generation DPCs(P10). Our findings demonstrate that ExoDSCCs(P3) promote the proliferation, viability and migration of senescent DPCs while inhibiting cell apoptosis. The expression of senescence marker SA-ß-Gal were significantly downregulated in senescent DPCs. When treated with ExoDSCCs(P3) , expression of inducibility related markers alkaline phosphatase and Versican were significantly upregulated. Additionally, ExoDSCCs(P3) activated the Wnt/ß-catenin signalling in vitro. In patch assay, ExoDSCCs(P3) significantly promoted hair follicle reconstruction in senescent DPCs. In summary, our work highlights that ExoDSCCs(P3) may restore the biological functions and improve the hair follicle induction ability of senescent DPCs. Therefore, ExoDSCCs(P3) may represent a new strategy for intervening in the ageing process of DPCs, contributing to the prevention of senile alopecia.

Fisetin, a potential skin rejuvenation drug that eliminates senescent cells in the dermis.

Accumulation of senescent fibroblasts, chronic inflammation, and collagen remodeling due to aging-related secretory phenotypes have been hypothesized to cause age-related skin aging, which results in wrinkles and loss of skin elasticity, thus compromising appearance attractiveness. However, the rejuvenating effects of removing senescent cells from the human skin and the efficacy of related therapeutic agents remain unclear. Here, we investigated the effects of fisetin, a potential anti-aging component found in various edible fruits and vegetables, on senescent human dermal fibroblasts (HDFs) and aging human skin. Senescence was induced in primary HDFs using long-term passaging and treatment with ionizing radiation, and cell viability was assessed after treatment with fisetin and a control component. A mouse/human chimeric model was established by subcutaneously transplanting whole skin grafts from aged individuals into nude mice, which were treated intraperitoneally with fisetin or control a component for 30 d. Skin samples were obtained and subjected to senescence-associated-beta-galactosidase staining; the extent of aging was evaluated using western blotting, reverse transcription-quantitative PCR, and histological analysis. Fisetin selectively eliminated senescent dermal fibroblasts in both senescence-induced cellular models; this effect is attributable to cell death induction by caspases 3, 8, and 9-mediated endogenous and exogenous apoptosis. Fisetin-treated senescent human skin grafts showed increased collagen density and decreased senescence-associated secretory phenotypes (SASP), including matrix metalloproteinases and interleukins. No apparent adverse events were observed. Thus, fisetin could improve skin aging through selective removal of senescent dermal fibroblasts and SASP inhibition, indicating its potential as an effective novel therapeutic agent for combating skin aging.

Age-related changes in dermal collagen physical properties in human skin.

Collagen is the major structural protein in the skin. Fragmentation and disorganization of the collagen fibrils are the hallmarks of the aged human skin dermis. These age-related alterations of collagen fibrils impair skin structural integrity and make the tissue microenvironment more prone to skin disorders. As the biological function of collagen lies predominantly in its physical properties, we applied atomic force microscopy (AFM) and nanoindentation to evaluate the physical properties (surface roughness, stiffness, and hardness) of dermal collagen in young (25±5 years, N = 6) and aged (75±6 years, N = 6) healthy sun-protected hip skin. We observed that in the aged dermis, the surface of collagen fibrils was rougher, and fiber bundles were stiffer and harder, compared to young dermal collagen. Mechanistically, the age-related elevation of matrix metalloproteinase-1 (MMP-1) and advanced glycation end products (AGEs) are responsible for rougher and stiffer/harder dermal collagen, respectively. Analyzing the physical properties of dermal collagen as a function of age revealed that alterations of the physical properties of collagen fibrils changed with age (22-89 years, N = 18). We also observed that the reticular dermis is rougher and mechanically stiffer and harder compared to the papillary dermis in human skin. These data extend the current understanding of collagen beyond biological entities to include biophysical properties.

Dermis resident macrophages orchestrate localized ILC2 eosinophil circuitries to promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4+ eosinophils, required to maintain their M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5+ type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5+ type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils.

Effects of oxytocin on the hair growth ability of dermal papilla cells.

Oxytocin (OXT) is a neuropeptide hormone termed "love hormone" produced and released during childbirth and lactation. It is also produced in response to skin stimulation (e.g., during hugging and massaging) and music therapy. The effects of OXT on various organs have been revealed in recent years; however, the relationship between hair follicles and OXT remains unclear. In this study, we examined the effects of OXT on dermal papilla (DP) cells that control hair growth by secreting growth/regression signals. Gene expression analysis revealed that DP signature markers were significantly upregulated in DP cells treated with OXT. In addition, we tested the hair growth-promoting effects of OXT using in vitro hair follicle organoids. OXT promoted the growth of hair peg-like sprouting by upregulating the expression of growth-promoting factors, including genes encoding vascular endothelial growth factor A (VEGFA). This study highlights the positive effects of OXT in hair follicles and may assist in the development of new treatments for alopecia.

Preparation of injectable porcine skin-derived collagen and its application in delaying skin aging by promoting the adhesion and chemotaxis of skin fibroblasts.

Collagen, as the main component of human skin, plays a vital role in maintaining dermal integrity. Its loss will lead to dermis destruction and collapse, resulting in skin aging. At present, injection of exogenous collagen is an important means to delay skin aging. In this study, high-purity collagen was extracted from porcine skin. Our research revealed that it can effectively promote the adhesion and chemotaxis of HSF cells. It can also reduce the expression of ß-galactosidase, decrease ROS levels, and increase the expression of the collagen precursors, p53 and p16 in HSF cells during senescence. After local injection into the aging skin of rats, it was found that the number of cells and type I collagen fibers in the dermis increased significantly, and the arrangement of these fibers became more uniform and orderly. Moreover, the important thing is that it is biocompatible. To sum up, the porcine skin collagen we extracted is an anti-aging biomaterial with application potential.

Identification of Glucocorticoid Receptor Target Genes That Potentially Inhibit Collagen Synthesis in Human Dermal Fibroblasts.

Over several decades, excess glucocorticoids (GCs) of endogenous or exogenous origin have been recognized to significantly inhibit collagen synthesis and accelerate skin aging. However, little is known regarding their molecular mechanisms. We hypothesized that the action of GCs on collagen production is at least partially through the glucocorticoid receptor (GR) and its target genes, and therefore aimed to identify GR target genes that potentially inhibit collagen synthesis in Hs68 human dermal fibroblasts. We first confirmed that dexamethasone, a synthetic GC, induced canonical GR signaling in dermal fibroblasts. We then collected 108 candidates for GR target genes reported in previous studies on GR target genes and verified that 17 genes were transcriptionally upregulated in dexamethasone-treated dermal fibroblasts. Subsequently, by individual knockdown of the 17 genes, we identified that six genes, AT-rich interaction domain 5B, FK506 binding protein 5, lysyl oxidase, methylenetetrahydrofolate dehydrogenase (NADP + dependent) 2, zinc finger protein 36, and zinc fingers and homeoboxes 3, are potentially involved in GC-mediated inhibition of collagen synthesis. The present study sheds light on the molecular mechanisms of GC-mediated skin aging and provides a basis for further research on the biological characteristics of individual GR target genes.

Evidence of inter- and intra-keloid heterogeneity through analysis of dermal fibroblasts: A new insight in deciphering keloid physiopathology.

Keloid scars are hypertrophic and proliferating pathological scars extending beyond the initial lesion and without tendency to regression. Usually, keloids are considered and treated as a single entity but clinical observations suggest heterogeneity in keloid morphologies with distinction of superficial/extensive and nodular entities. Within a keloid, heterogeneity could also be detected between superficial and deep dermis or centre and periphery. Focusing on fibroblasts as main actors of keloid formation, we aimed at evaluating intra- and inter-keloid fibroblast heterogeneity by analysing their gene expression and functional capacities (proliferation, migration, traction forces), in order to improve our understanding of keloid pathogenesis. Fibroblasts were obtained from centre, periphery, papillary and reticular dermis from extensive or nodular keloids and were compared to control fibroblasts from healthy skin. Transcriptional profiling of fibroblasts identified a total of 834 differentially expressed genes between nodular and extensive keloids. Quantification of ECM-associated gene expression by RT-qPCR brought evidence that central reticular fibroblasts of nodular keloids are the population which synthesize higher levels of mature collagens, TGFß, HIF1α and αSMA as compared to control skin, suggesting that this central deep region is the nucleus of ECM production with a centrifuge extension in keloids. Although no significant variations were found for basal proliferation, migration of peripheral fibroblasts from extensive keloids was higher than that of central ones and from nodular cells. Moreover, these peripheral fibroblasts from extensive keloids exhibited higher traction forces than central cells, control fibroblasts and nodular ones. Altogether, studying fibroblast features demonstrate keloid heterogeneity, leading to a better understanding of keloid pathophysiology and treatment adaptation.

EGFR marks a subpopulation of dermal mesenchymal cells highly expressing IGF1 which enhances hair follicle regeneration.

The skin harbours transcriptionally and functionally heterogeneous mesenchymal cells that participate in various physiological activities by secreting biochemical cues. In this study, we aimed to identify a new subpopulation of dermal mesenchymal cells that enhance hair follicle regeneration through a paracrine mechanism. Integrated single-cell RNA sequencing (scRNA-seq) data analysis revealed epidermal growth factor receptor (EGFR) as a marker of distinct fibroblast subpopulation in the neonatal murine dermis. Immunofluorescence staining and fluorescence-activated cell sorting (FACS) were used to validate the existence of the cell population in Krt14-rtTA-H2BGFP mouse. The difference of gene expression between separated cell subpopulation was examined by real-time PCR. Potential effect of the designated factor on hair follicle regeneration was observed after the application on excisional wounds in Krt14-rtTA-H2BGFP mouse. Immunofluorescence staining demonstrated the existence of dermal EGFR+ cells in neonatal and adult mouse dermis. The EGFR+ mesenchymal population, sorted by FACS, displayed a higher expression level of Igf1 (insulin-like growth factor 1). Co-localisation of IGF1 with EGFR in the mouse dermis and upregulated numbers of hair follicles in healed wounds following the application of exogenous IGF1 illustrated the contribution of EGFR+ cells in promoting wound-induced hair follicle neogenesis. Our results indicate that EGFR identifies a subpopulation of dermal fibroblasts that contribute to IGF1 promotion of hair follicle neogenesis. It broadens the understanding of heterogeneity and the mesenchymal cell function in skin and may facilitate the potential translational application of these cells.

Innovative Approaches and Advances for Hair Follicle Regeneration.

Pathological hair loss (also known as alopecia) and shortage of hair follicle (HF) donors have posed an urgent requirement for HF regeneration. With the revelation of mechanisms in tissue engineering, the proliferation of HFs in vitro has achieved more promising trust for the treatments of alopecia and other skin impairments. Theoretically, HF organoids have great potential to develop into native HFs and attachments such as sweat glands after transplantation. However, since the rich extracellular matrix (ECM) deficiency, the induction characteristics of skin-derived cells gradually fade away along with their trichogenic capacity after continuous cell passaging in vitro. Therefore, ECM-mimicking support is an essential prelude before HF transplantation is implemented. This review summarizes the status of providing various epidermal and dermal cells with a three-dimensional (3D) scaffold to support the cell homeostasis and better mimic in vivo environments for the sake of HF regeneration. HF-relevant cells including dermal papilla cells (DPCs), hair follicle stem cells (HFSCs), and mesenchymal stem cells (MSCs) are able to be induced to form HF organoids in the vitro culture system. The niche microenvironment simulated by different forms of biomaterial scaffold can offer the cells a network of ordered growth environment to alleviate inductivity loss and promote the expression of functional proteins. The scaffolds often play the role of ECM substrates and bring about epithelial-mesenchymal interaction (EMI) through coculture to ensure the functional preservation of HF cells during in vitro passage. Functional HF organoids can be formed either before or after transplantation into the dermis layer. Here, we review and emphasize the importance of 3D culture in HF regeneration in vitro. Finally, the latest progress in treatment trials and critical analysis of the properties and benefits of different emerging biomaterials for HF regeneration along with the main challenges and prospects of HF regenerative approaches are discussed.

Gellan gum-based hydrogels support the recreation of the dermal papilla microenvironment.

The dermal papilla (DP), a specialized compartment within the hair follicle, regulates hair growth. However, human DP cells rapidly lose their inductivity in 2D-culture given the loss of positional and microenvironmental cues. Spheroids have been capable of recreating the 3D intercellular organization of DP cells, however, DP cell-matrix interactions are poorly represented. Considering the specific nature of the DP's extracellular matrix (ECM), we functionalized gellan gum (GG) with collagen IV-(HepIII) or fibronectin-(cRGDfC) derived peptide sequences to generate a 3D environment in which the phenotype and physiological functions of DP cells are restored. We further tuned the stiffness of the microenvironments by varying GG amount. Biomimetic peptides in stiffer hydrogels promoted the adhesion of DP cells, while each peptide and amount of polymer independently influenced the type and quantity of ECM proteins deposited. Furthermore, although peptides did not seem to have an influence, stiffer hydrogels improved the inductive capacity of DP cells after short term culture. Interestingly, independently of the peptide, these hydrogels supported the recapitulation of basic hair morphogenesis-like events when incorporated in an organotypic human skin in vitro model. Our work demonstrates that tailored GG hydrogels support the generation of a microenvironment in which both cell-ECM and cell-cell interactions positively influence DP cells towards the creation of an artificial DP.

Possible Mechanisms of Stiffness Changes Induced by Stiffeners and Softeners in Catch Connective Tissue of Echinoderms.

The catch connective, or mutable collagenous, tissue of echinoderms changes its mechanical properties in response to stimulation. The body wall dermis of sea cucumbers is a typical catch connective tissue. The dermis assumes three mechanical states: soft, standard, and stiff. Proteins that change the mechanical properties have been purified from the dermis. Tensilin and the novel stiffening factor are involved in the soft to standard and standard to stiff transitions, respectively. Softenin softens the dermis in the standard state. Tensilin and softenin work directly on the extracellular matrix (ECM). This review summarizes the current knowledge regarding such stiffeners and softeners. Attention is also given to the genes of tensilin and its related proteins in echinoderms. In addition, we provide information on the morphological changes of the ECM associated with the stiffness change of the dermis. Ultrastructural study suggests that tensilin induces an increase in the cohesive forces with the lateral fusion of collagen subfibrils in the soft to standard transition, that crossbridge formation between fibrils occurs in both the soft to standard and standard to stiff transitions, and that the bond which accompanies water exudation produces the stiff dermis from the standard state.

Correlation between nuclear expression of heat shock protein 90 in dermis and glucocorticoid resistance in bullous dermatosis.

BACKGROUND: bullous dermatosis is a group of skin diseases that occur on the skin and mucous membrane, with blister and bulla as basic damage, mainly including pemphigus and bullous pemphigoid. Glucocorticoid (GC) is still the preferred drug for its treatment, but some patients respond poorly to GC and even develop glucocorticoid resistance (GCR). However, at present about the disease the understanding of the mechanisms for GCR is limited. OBJECTIVE: This study attempted to investigate the molecular mechanism of GCR in bullous dermatosis with heat shock proteins 90 (HSP90) and glucocorticoid receptor (GR) as molecular targets. METHODS: In this study, flow cytometry was used to measure and analyze the expression of HSP90 and GR in the lesions of patients with glucocorticoid-resistant bullosa dermatosis. Immunohistochemistry and immunofluorescence were used to observe the expression distribution and cell localization of HSP90 and GR. RESULTS: The expression of HSP90 in skin lesions of GCR group was significantly higher than that of glucocorticoid-sensitive (GCS) group, while the expression level of GR was lower than that of GCS group. In the epidermis, the expression and distribution of HSP90 were not different between the GCR group and the GCS group. And in the dermis, HSP90 and GR were more likely to be expressed in the nucleus in the GCR group. CONCLUSION: The overexpression and nuclear distribution of HSP90 may be related to the occurrence of GCR in patients with bullous dermatosis. And this correlation is more likely to occur in the dermis than in the epidermis.

The Epidermal Environment's Influence on the Dermal Environment in Response to External Stress.

INTRODUCTION: The outermost layer of the skin, the epidermis, is directly exposed to external stress (e.g., irradiation, allergens, and chemicals). Changes in epidermal conditions/environment in response to this stress could also influence conditions of the dermis, located directly beneath the epidermis. Yet, whether/how any epidermal environment changes in response to external stress affect dermal functions has not been completely clarified. METHODS: We employed ultraviolet irradiation B (UVB) (which hardly reaches the dermis) as a model of external stress. Human keratinocytes and human dermal fibroblasts were treated with UVB and conditioned medium of keratinocytes exposed to UVB (UVB-keratinocyte-M), respectively. We assessed (1) inflammatory cytokines and lipid mediators in keratinocytes; (2) matrix metalloprotease (MMP) levels and collagen degradation in fibroblasts; (3) ex vivo organ-cultured human skin was treated with UVB. MMP levels and collagen degradation were examined; (4) test whether the mixture of agent (agent cocktail) consisting of dihydroceramide, niacin amide, resveratrol, glucosyl hesperidin, and phytosterol ester that has been shown to improve skin barrier integrity can mitigate influence of UVB in skin; and (5) a pilot one-arm human clinical test to assess efficacy of formulation containing agent cocktail on stratum corneum hydration, skin elasticity, and wrinkle index. RESULTS: Inflammatory-cytokine and -lipid mediator production were increased in cultured keratinocytes treated with UVB, while matrix MMP-1, -3, and -9 production and collagen degradation were increased in fibroblasts incubated with UVB-keratinocyte-M. mRNA expression of COL1A1 (that codes type 1 collagen) levels was decreased in fibroblasts incubated with UVB-keratinocyte-M. The study using ex vivo organ-cultured human skin showed both MMP-1 and MMP-9 expression were increased in both epidermis and dermis and increased dermal collagen degradation following UVB irradiation. Increased MMP production and collagen degradation were attenuated by application of an agent cocktail. Finally, a pilot clinical study demonstrated that the formulation containing our agent cocktail likely has the ability to improve skin hydration, increase skin elasticity, and reduce the appearance of wrinkles. CONCLUSION: Epidermal changes in epidermal environment and conditions in response to external stress affect dermal conditions, and these negative effects of external stress on various skin layers can be pharmacologically mitigated.