SASP-induced macrophage dysfunction may contribute to accelerated senescent fibroblast accumulation in the dermis.

Recently, increasing attention has been paid to senescence-associated secretory phenotype (SASP), a phenomenon that senescent cells secrete molecules such as inflammatory cytokines and matrix metalloproteinases (MMPs), due to its noxious effects on the surrounding tissue. Senescent cells in the blood and liver are known to be properly depleted by macrophages. In the dermis, accumulation of senescent cells has been reported and is thought to be involved with skin ageing. In this study, to elucidate the clearance mechanism of senescent cells in the dermis, we focused on macrophage functions. Our co-culture experiments of senescent fibroblasts and macrophages revealed a two-step clearance mechanism: first, TNF-α secreted from macrophages induces apoptosis in senescent fibroblasts, and then, dead cells are phagocytosed by macrophages. Furthermore, it was suggested that SASP factors suppress both of the two steps of the senescent cell clearance by macrophages. From these findings, normally senescent cells in the dermis are thought to be removed by macrophages, but when senescent cells are excessively accumulated owing to oxidative stress, ultraviolet (UV) ray or other reasons, SASP was suggested to suppress the macrophage-dependent clearance functions and thereby cause further accumulation of senescent cells.

Increase of gremlin 2 with age in human adipose-derived stromal/stem cells and its inhibitory effect on adipogenesis.

INTRODUCTION: Adipose-derived stromal/stem cells (ASCs) have attracted attention as a promising material for regenerative medicine. Previously, we reported an age-related decrease in the adipogenic potential of ASCs from human subjects and found that the individual difference in this potential increased with age, although the mechanisms remain unclear. Recently, other groups demonstrated that a secreted antagonist of bone morphogenetic protein (BMP) signaling, Gremlin 2 (GREM2), inhibits the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into osteoblasts and the adipogenesis of 3T3-L1 cell. Here, we examined the effects of GREM2 on the differentiation of ASCs into adipocytes. METHODS: To examine changes in GREM2 expression levels with age, immunohistochemistry was performed on subcutaneous adipose tissues from subjects 12-97 years of age. Next, GREM2 gene expression levels in ASCs collected from subjects 5-90 years of age were examined by RT-PCR, and the change with age and correlation between the expression level and the adipogenic potential of ASCs were analyzed. In addition, to assess whether GREM2 affects adipogenesis, ASCs (purchased from a vendor) were cultured to induce adipogenesis with recombinant GREM2 protein, and siRNA-induced GREM2 knockdown experiment was also performed using aged ASCs. RESULTS: In adipose tissues, GREM2 expression was observed in cells, including ASCs, but not in mature adipocytes, and the expression level per cell increased with age. GREM2 expression levels in ASCs cultured in vitro also increased with age, and the individual differences in the level increased with age. Of note, partial correlation analysis controlled for age revealed that the adipogenic potential of ASCs and the GREM2 gene expression level were negatively correlated. Furthermore, based on a GREM2 addition experiment, GREM2 has inhibitory effects on the adipogenesis of ASCs through activation of Wnt/ß-catenin signaling. On the other hand, GREM2 knockdown in aged ASCs promoted adipogenesis. CONCLUSIONS: The GREM2 expression level was confirmed to play a role in the age-related decrease in adipogenic potential observed in ASCs isolated from adipose tissues as well as in the enhancement of the individual difference, which increased with age. GREM2 in adipose tissues increased with age, which suggested that GREM2 functions as an inhibitory factor of adipogenesis in ASCs.

UV irradiation-induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines.

Wnt/ß-catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/ß-catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5-carbon of cytosine (5mC), that is 5-methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non-SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non-SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non-SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.

CXCL12 regulates differentiation of human immature melanocyte precursors as well as their migration.

Melanocyte stem cells (McSCs) are localized in the bulge region of hair follicles and supply melanocytes, which determine hair color by synthesizing melanin. Ectopic differentiation of McSCs, which are usually undifferentiated in the bulge region, causes depletion of McSCs and results in hair graying. Therefore, to prevent hair graying, it is essential to maintain McSCs in the bulge region, but the mechanism of McSC maintenance remains unclear. To address this issue, we investigated the role of CXCL12, a chemokine which was previously suggested to induce migration of melanocyte lineage cells, as a niche component of McSCs. Immunohistological analysis revealed that CXCL12 was highly expressed in the bulge region of human hair follicles. CXCL12 mRNA expression level was significantly lower in white hairs plucked from human scalps than in black hairs. CXCL12 attracted the migration of early-passage normal human epidermal melanocytes (eNHEMs), an in vitro model of McSCs, which had characteristics of immature melanocyte precursors. We also found that CXCL12 suppressed their differentiation. These results suggest that CXCL12 regulates differentiation of McSCs as well as their proper localization, and maintaining McSCs by regulating CXCL12 expression level in the bulge region may be a key to preventing hair graying.

Extracellular proteoglycan decorin maintains human hair follicle stem cells.

Hair follicle stem cells (HFSC) are localized in the bulge region of the hair follicle and play a role in producing hair. Recently, it has been shown that the number of HFSC decreases with age, which is thought to be a cause of senile alopecia. Therefore, maintaining HFSC may be key for the prevention of age-related hair loss, but the regulatory mechanisms of HFSC and the effects of aging on them are largely unknown. In general, stem cells are known to require regulatory factors in the pericellular microenvironment, termed the stem cell niche, to maintain their cell function. In this study, we focused on the extracellular matrix proteoglycan decorin (DCN) as a candidate factor for maintaining the human HFSC niche. Gene expression analysis showed that DCN was highly expressed in the bulge region. We observed decreases in DCN expression as well as the number of KRT15-positive HFSC with age. In vitro experiments with human plucked hair-derived HFSC revealed that HFSC lost their undifferentiated state with increasing passages, and prior to this change a decrease in DCN expression was observed. Furthermore, knockdown of DCN promoted HFSC differentiation. In contrast, when HFSC were cultured on DCN-coated plates, they showed an even more undifferentiated state. From these results, as a novel mechanism for maintaining HFSC, it was suggested that DCN functions as a stem cell niche component, and that the deficit of HFSC maintenance caused by a reduction in DCN expression could be a cause of age-related hair loss.

Development of 3D imaging technique of reconstructed human epidermis with immortalized human epidermal cell line.

The epidermis, the outermost layer of the skin, retains moisture and functions as a physical barrier against the external environment. Epidermal cells are continuously replaced by turnover, and thus to understand in detail the dynamic cellular events in the epidermis, techniques to observe live tissues in 3D are required. Here, we established a live 3D imaging technique for epidermis models. We first obtained immortalized human epidermal cell lines which have a normal differentiation capacity and fluorescence-labelled cytoplasm or nuclei. The reconstituted 3D epidermis was prepared with these lines. Using this culture system, we were able to observe the structure of the reconstituted epidermis live in 3D, which was similar to an in vivo epidermis, and evaluate the effect of a skin irritant. This technique may be useful for dermatological science and drug development.

Laminin-332 regulates differentiation of human interfollicular epidermal stem cells.

Interfollicular epidermal stem cells (IFE-SCs) have self-renewal and differentiation potentials, and maintain epidermal homeostasis. Stem cells in vivo are regulated by the surrounding environment called niche to function properly, however, IFE-SC niche components are not fully understood. In order to elucidate the mechanisms of keeping epidermal homeostasis and of skin aging, and also to develop new therapeutic technologies for skin diseases, we searched for niche factors that regulate IFE-SCs. We found that laminin-332, a basement membrane component, was highly expressed at the tips of the dermal papillae, where IFE-SCs are localized, and that the stem cells by themselves expressed laminin-332. Knockdown of laminin-332 during the culture of IFE-SC-model cells to construct 3-dimensional epidermis in vitro resulted in failure to form proper structure, although no significant change was observed in either cell growth or apoptosis. Pre-coating of the culture insert with laminin-332 restored the normal formation of 3-dimensional epidermis. From these results, it was shown that laminin-332 is an essential niche component for the proper differentiation of IFE-SCs.

Interstitial fluid flow-induced growth potential and hyaluronan synthesis of fibroblasts in a fibroblast-populated stretched collagen gel culture.

BACKGROUND: Tensioned collagen gels with dermal fibroblasts (DFs) as a dermis model are usually utilized in a static culture (SC) that lacks medium flowing. To make the model closer to its in vivo state, we created a device to perfuse the model with media flowing at a physiological velocity and examined the effects of medium flow (MF) on the cultures. METHODS: We constructed a medium perfusion device for human DF-embedded stretched collagen gels (human dermis model), exposed the model to media that flows upwardly at ~1mL/day, and examined water retention of the gels, cells' growth ability, metabolic activity, expression profiles of nine extracellular matrix (ECM)-related genes. The obtained data were compared with those from the model in SC. RESULTS: MF increases the gels' water retention and cells' growth potential but had little effect on their metabolic activities. MF robustly enhanced hyaluronan synthase 2 (HAS2) and matrix metalloprotease 1 (MMP1) gene expressions but not of the other genes (MMP2, HYAL1, HYAL2, HYAL3, COL1A1, COL3A1, and CD44). MF significantly increased the amounts of cellular hyaluronan and adenosine triphosphate. CONCLUSIONS: The MF at a physiological speed significantly influences the nature of ECMs and their resident fibroblasts and remodels ECMs by regulating hyaluronan metabolism. GENERAL SIGNIFICANCE: Fibroblasts in tensioned collagen gels altered their phenotypes in a MF rate-dependent manner. Collagen gel culture with tension and MF could be utilized as an appropriate in vitro model of interstitial connective tissues to evaluate the pathophysiological significance of mechanosignals generated by fluid flow and cellular/extracellular tension.

Dermal CD271+ Cells are Closely Associated with Regeneration of the Dermis in the Wound Healing Process.

Stem cells have recently been shown to play important roles in wound healing. The aim of this study was to investigate the role of dermal CD271+ cells in wound healing. Full-thickness wounds were produced on the backs of 5-year-old and 24-week-old mice, and time-course of wound closure, CD271+ cell counts, and gene expression levels were compared. Delayed wound healing was observed in 24-week-old mice. The peak of CD271+ cell increase was delayed in 24-week-old mice, and gene expression levels of growth factors in wounded tissue were significantly increased in 5-year-old mice. Dermal CD271+ cells purified by fluorescence-activated cell sorting (FACS) expressed higher growth factors than CD271- cells, suggesting that CD271+ cells play important roles by producing growth factors. This study also investigated dermal CD271+ cells in patients with chronic skin ulcers. Dermal CD271+ cells in patients were significantly reduced compared with in healthy controls. Thus, dermal CD271+ cells are closely associated with wound healing.

Enhancement of individual differences in proliferation and differentiation potentials of aged human adipose-derived stem cells.

BACKGROUND: Adipose-derived stem cells (ASCs) are a robust, multipotent cell source. They are easily obtained and hold promise in many regenerative applications. It is generally considered that the function of somatic stem cells declines with age. Although several studies have examined the effects of donor age on proliferation potential and pluripotency of ASCs, the results of these studies were not consistent. OBJECTIVE: This study tested whether the donor age affects the yield of ASCs from adipose tissue, as well as the proliferation and differentiation potentials of ASCs. METHODS: This study used ASCs obtained from adipose tissues of 260 donors (ages 5-97 years). ASCs were examined for individual differences in proliferation, and adipogenic, osteogenic and chondrogenic differentiation potentials in vitro. Characteristics of ASCs from each donor were evaluated by the principal component analysis (PCA) using their potential parameters. RESULTS: Analyses on ASCs demonstrated that adipogenic potentials declined with age, but proliferation, osteogenic and chondrogenic potentials were not correlated with age. Interestingly, in all ASC potentials, including adipogenesis, individual differences were observed. Principal component analysis (PCA) revealed that individual differences became evident in the elderly, and those variations were more prominent in females than in males. CONCLUSIONS: This study demonstrated age-related changes in the potentials of ASCs and revealed that the individual differences of ASCs become significant in people over 60 years of age (for females over 60, and for males over 80). We believe that it is important to carefully observe ASC potentials in order to achieve effective regenerative medicine treatments using ASCs.

Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

Inhibitory effect of Phalaenopsis orchid extract on WNT1-induced immature melanocyte precursor differentiation in a novel in vitro solar lentigo model.

Recently, it has been reported that increased expression of WNT1 accelerates the differentiation of melanocyte stem cells (McSCs) in solar lentigines (SLs), hyperpigmented maculae commonly seen on sun-exposed areas of the skin. In this study, to establish an in vitro SL model, human epidermal squamous carcinoma cell line HSC-1, which expresses higher levels of WNT1 than normal human epidermal keratinocytes, was co-cultured with early passage normal human epidermal melanocytes (NHEMs) as an in vitro McSC model. As a result, mRNA expression levels of melanocyte differentiation-related genes MITF and TYR in NHEMs were significantly increased by co-culturing with HSC-1 cells. Furthermore, Phalaenopsis orchid extract (Phex) inhibited McSCs differentiation by suppressing WNT1 expression via down-regulation of DLX2, a transcriptional activator of WNT1, in HSC-1 cells. Therefore, our finding suggested that extracts such as Phex, which suppresses WNT1 expression, may be useful as a novel treatment of SLs.

Microorganisms inhabiting follicular contents of facial acne are not only Propionibacterium but also Malassezia spp.

To clarify the relationship between major cutaneous microorganisms (Propionibacterium, Staphylococcus and Malassezia spp.) and acne vulgaris (acne), we examined the microbiota quantitatively in the follicular contents of inflammatory acne and on the facial skin of patients with acne. Fifteen Japanese untreated acne outpatients were studied. The follicular contents from inflammatory acne lesions of the face were collected using a comedo extractor. The skin surface samples were obtained by the swab method from 10 cm(2) of facial skin. The microbiota was analyzed using polymerase chain reaction. The microbiota in follicular contents was similar to that on the skin surface, namely, there were large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. Moreover, the number of Malassezia spp. on the skin surface was correlated with that of inflammatory acne and that in follicular contents. This study clarified that there are large populations of Propionibacterium spp., Staphylococcus spp. and Malassezia spp. in follicular contents. These results suggest the possibility that not only Propionibacterium acnes but also other cutaneous resident microorganisms are related to acne. Particularly, we considered that Malassezia spp. is closely related.

Age-related decrease in CD271(+) cells in human skin.

According to recent studies, stem cells are found in various tissues in our bodies. It has been reported that stem cells can reside in the skin tissues, including the epidermis, dermis, hair follicles and subcutaneous tissues. Homeostasis of the skin is maintained because these stem cells collaborate with each other to form new cells. We previously identified the CD271(p75NTR)(+) cell as a stem cell that was present in the epidermis, dermis and subcutaneous tissue, and further investigated the role of stem cells in wound healing and their association with skin disease. In this study, we investigated the localization of CD271(+) cells in human skin (epidermis and dermis) and its age-related changes in stem cells using CD271(+) cells. The study revealed that the number of CD271(+) cells in the epidermis and dermis decreased with aging. It is possible that such an age-related decrease in stem cells causes impaired regenerative ability and is associated with various skin diseases. If the relationship between stem cells and skin aging and diseases can be elucidated by investigations such as this study, it may lead to the development of novel anti-aging technologies and medical treatments for skin diseases in the future.

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis.

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.

Fatty acid compositions of triglycerides and free fatty acids in sebum depend on amount of triglycerides, and do not differ in presence or absence of acne vulgaris.

To clarify the influence of the fatty acid composition of sebum in acne vulgaris, we investigated the amounts and fatty acid compositions of triglycerides (TG) and free fatty acids (FFA), and the amounts of cutaneous superficial Propionibacterium acnes in acne patients and healthy subjects. The foreheads of 18 female patients, 10 male patients, 10 healthy females and 10 healthy males were studied in a Japanese population. There were significant differences in the amounts of sebum, TG and cutaneous superficial P. acnes, as well as the fatty acid compositions of TG and FFA between acne patients and healthy subjects in females. Their fatty acid compositions were correlated with the amount of TG with or without acne. It was clarified that the fatty acid compositions of TG and FFA depended on the amount of TG, and there were no differences in the fatty acid composition in the presence and absence of acne.

Increase of stratifin triggered by ultraviolet irradiation is possibly related to premature aging of human skin.

Although ultraviolet (UV) rays cause premature aging of human skin, which is called photoaging, its detailed mechanisms are not known. Stratifin (SFN), a member of the 14-3-3 protein family, is secreted by keratinocytes on human skin, and has an effect on gene expression in other cells. In this study, the association of SFN with the mechanism of photoaging was investigated. The effect of UVB irradiation on SFN expression in epidermal keratinocytes was examined by in vitro and in vivo studies. In addition, the effects of SFN on epidermal keratinocytes and dermal fibroblasts were examined. SFN mRNA expression and protein levels increased significantly in UVB-irradiated keratinocytes. SFN significantly decreased filaggrin and serine palmitoyltransferase mRNA expression in epidermal keratinocytes and hyaluronan synthase 2 mRNA expression in dermal fibroblasts. In addition, it was reconfirmed that SFN induces the downregulation of collagen content through changes of COL-1, MMP-1 and MMP-2 mRNA expressions. Furthermore, the expression level of SFN mRNA was significantly higher in sun-exposed compared with that in sun-shielded skin. These results suggest that SFN affects the water-holding capacity, barrier function and dermal matrix components in photoaging skin. An increase of SFN triggered by UVB irradiation may be one of the causes of alterations observed in photoaging skin.

Accelerated differentiation of melanocyte stem cells contributes to the formation of hyperpigmented maculae.

It has been reported that the abnormal regulation of melanocyte stem cells (McSCs) causes hair greying; however, little is known about the role of McSCs in skin hyperpigmentation such as solar lentigines (SLs). To investigate the involvement of McSCs in SLs, the canonical Wnt signalling pathway that triggers the differentiation of McSCs was analysed in UVB-induced delayed hyperpigmented maculae in mice and human SL lesions. After inducing hyperpigmented maculae on dorsal skin of F1 mice of HR-1× HR/De, which was formed long after repeated UVB irradiation, the epidermal Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were increased as compared to non-irradiated control mice. Furthermore, the expression of dopachrome tautomerase (Dct), a downstream target of ß-catenin, was significantly upregulated in McSCs of UVB-irradiated mice. The Wnt1 expression and the number of nuclear ß-catenin-positive McSCs were also higher in human SL lesions than in normal skin. Recombinant Wnt1 protein induced melanocyte-related genes including Dct in early-passage normal human melanocytes (NHEMs), an in vitro McSC model. These results demonstrate that the canonical Wnt signalling pathway is activated in SL lesions and strongly suggest that the accelerated differentiation of McSCs is involved in SL pathogenesis.

ZIP2 protein, a zinc transporter, is associated with keratinocyte differentiation.

Zinc is essential for the proper functioning of various enzymes and transcription factors, and its homeostasis is rigorously controlled by zinc transporters (SLC39/ZIP, importers; SLC30/ZnT, exporters). Skin disease is commonly caused by a zinc deficiency. Dietary and inherited zinc deficiencies are known to cause alopecia and the development of vesicular or pustular dermatitis. A previous study demonstrated that zinc played crucial roles in the survival of keratinocytes and their unique functions. High levels of zinc have been detected in the epidermis. Epidermal layers are considered to use a mechanism that preferentially takes in zinc, which is involved with the unique functions of keratinocytes. However, few studies have investigated the ZIP (Zrt- and Irt-like protein) proteins specifically expressed in keratinocytes and their functions. We explored the ZIP proteins specifically expressed in the epidermis and analyzed their functions. Gene expression analysis showed that the expression of ZIP2 was consistently higher in the epidermis than in the dermis. Immunohistochemistry analysis confirmed the expression of ZIP2 in differentiating keratinocytes. The expression of ZIP2 was found to be up-regulated by the differentiation induction of cultured keratinocytes. Intracellular zinc levels were decreased in keratinocytes when ZIP2 was knocked down by siRNA, and this subsequently inhibited the differentiation of keratinocytes. Moreover, we demonstrated that ZIP2 knockdown inhibited the normal formation of a three-dimensional cultured epidermis. Taken together, the results of this study suggest that ZIP2, a zinc transporter expressed specifically in the epidermis, and zinc taken up by ZIP2 are necessary for the differentiation of keratinocytes.