Ingestion of Coffee Polyphenols Improves a Scaly Skin Surface and the Recovery Rate of Skin Temperature after Cold Stress: A Randomized, Controlled Trial.

Coffee polyphenols (CPPs) derived from coffee beans have beneficial effects on blood pressure and vascular endothelial function. In addition, CPPs suppress ultraviolet light induced erythema. However, the effects of CPPs on dry skin and cutaneous vascular function have not been clarified. We investigated the effects of CPPs on dry skin and the recovery rate (RR) of skin temperature after a cold-stress test as a measure of vascular function in subjects with visible scaliness in a double-blind, placebo-controlled, randomized study. The subjects were divided into two groups, the CPP group and the Placebo group. In the CPP group, the subjects ingested a beverage containing 297.8 mg CPPs every day for 4 wk. The degree of skin dryness was assessed quantitatively using a Visioscan to evaluate skin scaliness and smoothness. A subjective evaluation using a visual analog scale (VAS) of skin smoothness was also used. As a result, the scaliness and smoothness of cheek skin was significantly improved after 4 wk in the CPP group compared to the Placebo group. The improvements of the VAS score on 'skin smoothness' and the RR were also observed in the CPP group but the difference was not statistically significant. However, when the CPP group was divided into subgroups of high RR and low RR, the improvement of the RR was significant in the low RR subgroup. In conclusion, our results suggest that CPPs improve skin scaliness and play a role in cutaneous blood flow regulation after cold stress.

Dry skin conditions are related to the recovery rate of skin temperature after cold stress rather than to blood flow.

BACKGROUND: Cutaneous blood flow plays an important role in the thermoregulation, oxygen supply, and nutritional support necessary to maintain the skin. However, there is little evidence for a link between blood flow and skin physiology. Therefore, we conducted surveys of healthy volunteers to determine the relationship(s) between dry skin properties and cutaneous vascular function. METHODS: Water content of the stratum corneum, transepidermal water loss, and visual dryness score were investigated as dry skin parameters. Cutaneous blood flow in the resting state, the recovery rate (RR) of skin temperature on the hand after a cold-stress test, and the responsiveness of facial skin blood flow to local cooling were examined as indices of cutaneous vascular functions. The relationships between dry skin parameters and cutaneous vascular functions were assessed. RESULTS: The RR correlated negatively with the visual dryness score of skin on the leg but correlated positively with water content of the stratum corneum on the arm. No significant correlation between the resting state of blood flow and dry skin parameters was observed. In both the face and the body, deterioration in skin dryness from summer to winter was significant in subjects with low RR. The RR correlated well with the responsiveness of facial skin blood flow to local cooling, indicating that the RR affects systemic dry skin conditions. CONCLUSIONS: These results suggest that the RR but not blood flow at the resting state is associated with dry skin conditions and is involved in skin homeostasis during seasonal environmental changes.

Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin.

Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-ß antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-ß-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process.

Immunomagnetic exclusion of PECAM-1-positive endothelial cells in fetal mouse liver cell cultures causes impaired growth and gene expression of hepatoblasts and stellate cells.

Previous studies using mice having defective VEGF signaling have demonstrated that vascular development is indispensable for early hepatic organogenesis. However, not only whether its action lasts during later hepatic development, but also what molecules are involved in that action remains to be determined. The present study was undertaken to examine the effects of primitive sinusoidal endothelial cells on hepatic growth and maturation in primary culture of fetal mouse liver cells, and to determine their molecular mechanisms. When endothelial cells were excluded from E12.5 liver cell cultures by using PECAM-1-antibody-coated magnetic beads, the growth of hepatoblasts and stellate cells was conspicuously reduced and hepatic maturation was also suppressed. Conditioned medium prepared from fetal liver cell cultures containing almost all hepatic cell types stimulated the growth and gene expression of hepatoblasts and stellate cells similarly to the cultures in the presence of endothelial cells. HGF mRNA expression was downregulated in endothelial cellfree cultures of fetal liver cells, and the addition of HGF to the culture medium rescued the cells from the effects of endothelial cell depletion. These data suggest that humoral factors, including HGF, which are produced by endothelial cells or stellate cells, are involved in fetal hepatocyte growth and maturation.

Analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes.

Aquaporin 9 (AQP9) is a member of the aquaglyceroporin family that transports glycerol, urea and other small solutes as well as water. Compared to the expression and function in epidermal keratinocytes of AQP3, another aquaglyceroporin, our knowledge of epidermal AQP9 remains elusive. In this study, we investigated the expression of AQP9 in the human epidermis and cultured keratinocytes. Immunofluorescence studies revealed that AQP9 expression is highly restricted to the stratum granulosum of the human epidermis, where occludin is also expressed at the tight junctions. Interestingly, the AQP3 staining decreased sharply below the cell layers in which AQP9 is expressed. In cultured normal human epidermal keratinocytes (NHEK), knock-down of AQP9 expression in the differentiated cells induced by RNA interference reduced glycerol uptake, which was not as pronounced as was the case with AQP3 knock-down cells. In contrast, similar reduction of urea uptake was detected in AQP9 and AQP3 knock-down cells. These findings suggested that AQP9 expression in NHEK facilitates at least the transport of glycerol and urea. Finally, we analyzed the effect of retinoic acid (RA), a potent stimulator of keratinocyte proliferation, on AQP3 and AQP9 mRNA expression in differentiated NHEK. Stimulation with RA at 1 µM for 24 h augmented AQP3 expression and down-regulated AQP9 expression. Collectively, these results indicate that AQP9 expression in epidermal keratinocytes is regulated in a different manner from that of AQP3.

N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.

Murine homologue of the human KIAA1199 is implicated in hyaluronan binding and depolymerization.

Recently, we have disclosed that human KIAA1199 (hKIAA1199) is a hyaluronan (HA) binding protein implicated in HA depolymerization. Although a murine homologue (mKiaa1199) was previously cloned, no information about the function of the molecule was available. Here, we show that cells transfected with mKiaa1199 cDNA selectively catabolized HA via the clathrin-coated pit pathway. A glycosaminoglycan-binding assay demonstrated the specific binding of mKiaa1199 to HA. These results were similar to our observations with hKIAA1199, although slight differences were found in the peak sizes of the minimum degradates of HA. We conclude that like hKIAA1199, mKiaa1199 is a hyaladherin, leading to HA depolymerization.

Lutein, a nonprovitamin A, activates the retinoic acid receptor to induce HAS3-dependent hyaluronan synthesis in keratinocytes.

Carotenoids have been reported to have potent antioxidant activities and to protect tissues and cells from certain diseases and environmental insults. The molecular mechanism of the action of provitamin A carotenoids such as ß-carotene and ß-cryptoxanthin is mediated in part by retinoic acid, an active form of provitamin A, but the molecular basis of the biological activities of non-provitamin A carotenoids such as lutein, zeaxanthin, and astaxanthin is not fully understood. In this study, we investigated to determine whether the actions of non-provitamin A carotenoids are mediated via retinoid signaling by monitoring retinoic acid receptor (RAR)-dependent hyaluronan production in cultured human keratinocytes. Not only ß-carotene and ß-cryptoxanthin, but also lutein, zeaxanthin, and astaxanthin, upregulated HAS3 gene expression and were followed by hyaluronan synthesis. We found that LE540, an antagonist of retinoic acid receptors, abolished lutein dependent hyaluronan synthesis and that lutein significantly increased retinoic acid responsive element (RARE)-driven transcript acitivity. In addition, we found that citral, an inhibitor of retinal dehydrogenases, decreased lutein-stimulated hyaluronan synthesis, indicating that lutein metabolites rather than lutein itself act as an RAR ligand in RAR-mediated transcription activity in keratinocytes. A series of non-provitamin A can be substituted for retinoids and should be considered as a potential means of improving skin health.

KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.

Hyaluronan (HA) has an extraordinarily high turnover in physiological tissues, and HA degradation is accelerated in inflammatory and neoplastic diseases. CD44 (a cell surface receptor) and two hyaluronidases (HYAL1 and HYAL2) are thought to be responsible for HA binding and degradation; however, the role of these molecules in HA catabolism remains controversial. Here we show that KIAA1199, a deafness gene of unknown function, plays a central role in HA binding and depolymerization that is independent of CD44 and HYAL enzymes. The specific binding of KIAA1199 to HA was demonstrated in glycosaminoglycan-binding assays. We found that knockdown of KIAA1199 abolished HA degradation by human skin fibroblasts and that transfection of KIAA1199 cDNA into cells conferred the ability to catabolize HA in an endo-ß-N-acetylglucosaminidase-dependent manner via the clathrin-coated pit pathway. Enhanced degradation of HA in synovial fibroblasts from patients with osteoarthritis or rheumatoid arthritis was correlated with increased levels of KIAA1199 expression and was abrogated by knockdown of KIAA1199. The level of KIAA1199 expression in uninflamed synovium was less than in osteoarthritic or rheumatoid synovium. These data suggest that KIAA1199 is a unique hyaladherin with a key role in HA catabolism in the dermis of the skin and arthritic synovium.

Impaired tight junctions obstruct stratum corneum formation by altering polar lipid and profilaggrin processing.

BACKGROUND: The stratum corneum (SC) is a well-known structure responsible for the cutaneous barrier. Tight junctions (TJs) function as a paracellular barrier beneath the SC and are involved in the cutaneous barrier. It remains unclear how TJs are involved in the cutaneous barrier. OBJECTIVE: In order to clarify the role of TJs in the cutaneous barrier, we investigated skin equivalent models with disrupted TJ barriers focusing on the SC. METHODS: Skin equivalents with disrupted TJ barriers were established using GST-C-CPE, a peptide with specific inhibitory action against specific claudins. The changes of the SC barrier in the skin equivalents with disrupted TJ barriers were investigated and compared with control skin equivalents. RESULTS: An outside-to-inside skin barrier assay revealed a defective SC barrier in skin equivalents with disrupted TJ barriers. A detailed examination of the SC revealed an increase in the pH of the SC in the skin equivalent with disrupted TJ barriers. An electron microscopy showed the failure of lamellar structures to mature and the failure of keratohyalin granules to degrade in the skin equivalents with disrupted TJ barriers. A thin layer chromatography analysis showed an increase in polar lipids and a decrease in non-polar lipids. A western blot analysis showed an increase in filaggrin dimer and trimer and a decrease in filaggrin monomer. CONCLUSION: We found that disrupted TJs obstructed the SC formation responsible for the cutaneous barrier. Our study indicates the possibility that impaired TJ barriers affect polar lipids and profilaggrin processing by disturbing the pH condition of the SC.

Chemokine-dependent T cell migration requires aquaporin-3-mediated hydrogen peroxide uptake.

Chemokine-dependent trafficking is indispensable for the effector function of antigen-experienced T cells during immune responses. In this study, we report that the water/glycerol channel aquaporin-3 (AQP3) is expressed on T cells and regulates their trafficking in cutaneous immune reactions. T cell migration toward chemokines is dependent on AQP3-mediated hydrogen peroxide (H(2)O(2)) uptake but not the canonical water/glycerol transport. AQP3-mediated H(2)O(2) transport is essential for the activation of the Rho family GTPase Cdc42 and the subsequent actin dynamics. Coincidentally, AQP3-deficient mice are defective in the development of hapten-induced contact hypersensitivity, which is attributed to the impaired trafficking of antigen-primed T cells to the hapten-challenged skin. We therefore suggest that AQP3-mediated H(2)O(2) uptake is required for chemokine-dependent T cell migration in sufficient immune response.

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells.

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes.

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.

Changes in epidermal hyaluronan metabolism following UVB irradiation.

BACKGROUND: Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation, and have shown different biological activities depending on its molecular mass. While many studies have shown changes in the amount of HA after UVB irradiation, molecular mass change remains to be elucidated. OBJECTIVE: To investigate the change in the molecular mass of HA after UVB irradiation in mouse epidermis. METHODS: The mice were irradiated with a single dose of UVB (0.15J/cm(2)). The amount of HA was examined using HABP sandwich assay. The molecular mass distribution was estimated by Sephacryl S-1000 chromatography. Has and Hyal mRNA expressions were detected by real-time PCR. RESULTS: On day 2 after UVB irradiation, both the amount of HA and the up-regulation of Has3 mRNA expression reached their maximum. The average HA molecular mass was about 1000 kDa, a level similar to that of the non-irradiated epidermis. On day 3, the average HA molecular mass drastically decreased to 100 kDa, while Hyal1, Hyal2, and Hyal3 mRNA expressions slightly increased. The amount of HA, however, remained high. On days 4 and 5, the amount of HA gradually decreased, but the molecular mass of HA remained low. A drastic reduction of the HA molecular mass after UVB irradiation was confirmed. CONCLUSION: UVB irradiation elicits remarkable changes in the molecular mass of HA, as well as amount. These qualitative and quantitative changes of HA might play an important role in UVB-induced cell proliferation and differentiation. Further study will be required to resolve the mechanism of HA degradation in the epidermis.

The presence of N(ε)-(Carboxymethyl) lysine in the human epidermis.

It is well known that advanced glycation end products (AGEs) are formed in long-lived dermal proteins such as collagen, and that their formation is related to skin aging. To examine the distribution of AGEs in skin tissue, we performed immunofluorescence studies on the human skin using an anti-AGEs antibody. Interestingly, AGEs signals were observed not only in the dermis but also in the epidermis. The objectives of this study were to confirm the presence of N(ε)-(Carboxymethyl) lysine (CML), an AGE structure, in the epidermis and to characterize the CML-modified proteins. The presence of CML in the stratum corneum (SC) was examined using liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Concordance between the retention times of a compound in the SC hydrolysate and authentic CML, as well as with the specific mass transition of CML, was detected. This result showed that CML is present in the epidermis. In order to characterize the CML-modified proteins in the epidermis, protein samples extracted from the SC were analyzed using two-dimensional electrophoresis followed by an amino acid sequence analysis. The clarified peptide sequences covered approximately 27% of the amino acid sequences of cytokeratin 10 (K10). In the immunoblotting experiment following the two-dimensional electrophoresis, where protein samples extracted from whole epidermis were used, the position of the major CML-positive spots corresponded to those of K10. Taken together these results showed that CML is present in the human epidermis, and suggest that K10 is one of the target molecules for CML modification in the epidermis.

Characterization of tight junctions and their disruption by UVB in human epidermis and cultured keratinocytes.

It has not been confirmed whether tight junctions (TJs) function as a paracellular permeability barrier in adult human skin. To clarify this issue, we performed a TJ permeability assay using human skin obtained from abdominal plastic surgery. Occludin, a marker protein of TJs, was expressed in the granular layer, in which a subcutaneously injected paracellular tracer, Sulfo-NHS-LC-Biotin (556.59 Da), was halted. Incubation with ochratoxin A decreased the expression of claudin-4, an integral membrane protein of TJs, and the diffusion of paracellular tracer was no longer prevented at the TJs. These results demonstrate that human epidermis possesses TJs that function as an intercellular permeability barrier at least against small molecules (∼550 Da). UVB irradiation of human skin xenografts and human skin equivalents (HSEs) resulted in functional deterioration of TJs. Immunocytochemical staining of cultured keratinocytes showed that occludin was localized into dot-like shapes and formed a discontinuous network when exposed to UVB irradiation. Furthermore, UVB irradiation downregulated the active forms of Rac1 and atypical protein kinase C, suggesting that their inactivation caused functional deterioration of TJs. In conclusion, TJs function as a paracellular barrier against small molecules (∼550 Da) in human epidermis and are functionally deteriorated by UVB irradiation.

Adiponectin resides in mouse skin and upregulates hyaluronan synthesis in dermal fibroblasts.

Adipose tissue is a hormonally active tissue that produces adipokines that influence the activity of other tissues. Adiponectin is an adipocyte-specific adipokine involved in systemic metabolism. We detected the expression of adiponectin receptors (AdipoR1 and AdipoR2) mRNA in cultured dermal fibroblasts. The full-length adiponectin (fAd), but not the globular adiponectin (gAd), increased hyaluronan (HA) production and upregulated HA synthase (HAS) 2 mRNA expression. AdipoR1 and AdipoR2 mRNAs were also expressed in keratinocytes, though neither fAd nor gAd had any effect on HA synthesis. In mouse skin, we found that adiponectin was present and decreased markedly with aging. The age-dependent pattern of adiponectin decrease in skin, correlated well with that of HA in skin. Our experiments were also the first to identify adiponectin production in cultured mouse sebocytes, a finding that suggests that skin adiponectin may derive not only from plasma and/or subcutaneous adipose tissue, but also from the sebaceous gland. These results indicated that adiponectin plays an important role in the HA metabolism of skin.

Developmental changes of cell adhesion molecule expression in the fetal mouse liver.

Developmental changes of cell adhesion molecule expression, especially in nonparenchymal cells, have hardly ever been analyzed in the murine liver. The present study was undertaken to immunohistochemically examine the expression of NCAM, ICAM, VCAM, and N-cadherin during mouse liver development and in fetal liver cell cultures. NCAM was transiently expressed in mesenchymal cells of the septum transversum and sinusoidal cells in liver development. In vitro studies demonstrated that desmin-positive stellate cells expressed this cell adhesion molecule. NCAM expression in periportal biliary epithelial cells and connective tissue cells also coincided well with bile duct remodeling processes in the perinatal periods. Expression of ICAM and VCAM was transiently restricted to hepatoblasts, hepatocytes and hemopoietic cells in fetal stages. N-cadherin was expressed not only in hepatoblasts and hepatocytes, but also in nonparenchymal cells such as endothelial cells, stellate cells and connective tissue cells, however the expression was weak. These results suggest that each cell adhesion molecule may play an important role during development in hepatic histogenesis, including hepatoblast/hepatocyte-stellate cell interactions, hemopoiesis, and bile duct morphogenesis.

XPA gene mutations resulting in subtle truncation of protein in xeroderma pigmentosum group A patients with mild skin symptoms.

Comparisons of the clinical manifestations with gene mutations in patients with xeroderma pigmentosum group A (XPA) have suggested that those with mutations closer to the C-terminal coding region of the XPA gene have milder neurological and cutaneous symptoms. Here we report on four middle-aged, newly diagnosed Japanese XPA patients whose unusually mild symptoms, especially those affecting the skin, implicate a reduced association of a subtle defect in the C-terminus of XPA protein with skin lesions. All patients had a heterozygous G → C transversion at the splice acceptor site of XPA intron 3. We identified previously unreported heterozygous mutations in exon 6: a single-base insertion (690insT) in one patient and a four-base insertion (779insTT and 780insTT) in the other patients. These mutations led to the frameshift that created new premature termination codons, resulting in the production of truncated XPA proteins. They were longer than any previously reported truncated XPA protein, suggesting that the minimal cutaneous symptoms in these patients are due to a higher residual level of XPA protein activity and that the subtle defect in the C-terminus of XPA protein is more closely related to neurological impairment than to cutaneous abnormalities.