Carbon dioxide ameliorates reduced desquamation in dry scaly skin via protease activation.

OBJECTIVE: Scaling, a phenomenon showing an abnormal detachment of the stratum corneum (SC) owing to desquamation dysfunction, is commonly observed in various skin diseases or xerotic skin due to ageing and low humidity. Therefore, it is considered that ameliorating the disturbed desquamatory process of the SC leads to improvement in scaling. Carbon dioxide (CO2 ) is known to be good for some skin diseases; however, the effect of CO2 on scaling and its mechanism are not sufficiently clear. We aimed to elucidate the effect of transepidermal application of CO2 on scaling and its mechanism of action. METHODS: Twenty healthy men with mild scaling on the cheeks were recruited for a double-blind, placebo-controlled, split-face study. They applied the formulation containing CO2 twice daily for 1 week. After the study, the SC was collected by tape stripping to analyse desquamatory protease activities and degradation of extracellular corneodesmosomes. Furthermore, the contribution of pH to proteolysis of the corneodesmosome by CO2 was evaluated using three-dimensional (3D) cultured epidermal models. RESULTS: The spectroscopic absorbance of tape strips, used as scaling indicators, was decreased, concomitantly with the amelioration of incomplete degradation of desmoglein-1, one of the main corneodesmosomal proteins, and activation of trypsin-like protease in the SC by transepidermal application of CO2 . Experiments using 3D cultured epidermis showed that pH in the epidermal tissue was lowered by CO2 , whereas a pH change was not observed with the application of the formulation containing hydrochloric acid, which was added to equalize the pH to that of the CO2 formulation. CONCLUSION: The transcutaneous application of CO2 ameliorates reduced desquamatory process in xerotic skin, with concomitant mild acidification of the SC, thereby leading to improvement in scaling. Thus, CO2 may have an advantage of efficiently and safely counteracting scaling of various skin disorders.

OBJECTIF: La desquamation, phénomène caractérisé par un détachement anormal de la couche cornée (CC) dû à un dysfonctionnement de l'épiderme, est fréquemment observée dans diverses maladies de la peau ou en cas de xérose résultant du vieillissement et de la faible humidité. Par conséquent, il est considéré que le soulagement du trouble à l'origine du processus desquamant de la CC réduit la desquamation. Le dioxyde de carbone (CO2) est réputé bénéfique pour certaines maladies de la peau. Cependant, l'effet du CO2 sur la desquamation et son mécanisme ne sont pas suffisamment clairs. Nous avons cherché à élucider l'effet de l'application transépidermique du CO2 sur la desquamation et son mécanisme d'action. MÉTHODES: Vingt hommes en bonne santé, présentant une légère desquamation sur les joues, ont été recrutés dans le cadre d'une étude en double aveugle, contrôlée par un placebo, en hémiface. Ils ont appliqué la formule contenant du CO2 deux fois par jour, pendant 1 semaine. Après l'étude, la CC a été recueillie par décollement de ruban adhésif, en vue de l'analyse des activités de la protéase desquamante et de la dégradation des cornéodesmosomes extracellulaires. En outre, la contribution du pH à la protéolyse du cornéodesmosome par le CO2 , a été évaluée à l'aide de modèles d'épidermes cultivés tridimensionnels (3D). RÉSULTATS: L'absorbance spectroscopique des bandelettes de ruban adhésif, utilisées comme indicateurs de desquamation, a été réduite, concomitamment avec la baisse de la dégradation incomplète de la desmogléine-1, l'une des principales protéines des cornéodesmosomes, et l'activation de la trypsine dans la CC par application transépidermique de CO2 . Des expériences menées sur un épiderme cultivé en 3D ont montré que le pH dans le tissu épidermique était réduit par le CO2 , tandis qu'aucun changement de pH n'a été observé avec l'application de la formule contenant de l'acide chlorhydrique, ajoutée pour que le pH soit identique à celui de la formule contenant du CO2 . CONCLUSION: L'application transcutanée de CO2 améliore la réduction du processus desquamant de la peau atteinte de xérose, avec une légère acidification concomitante de la CC, entraînant ainsi une réduction de la desquamation. Par conséquent, le CO2 peut présenter l'avantage de contrer la desquamation de manière efficace et sûre, pour diverses affections cutanées.

Correction to: Characterization of skin function associated with obesity and specific correlation to local/systemic parameters in American women.

Following publication of the original article [1], the authors identified an error. In the description in Fig. 1b the "solid line" "dashed line" should be exchanged. The original article has been updated.

Characterization of skin function associated with obesity and specific correlation to local/systemic parameters in American women.

BACKGROUND: Obesity is considered problematic not only as a major cause of diabetes, hypertension, and dyslipidemia, but also as a risk of intractable dermatosis; however influence of obesity on skin function has not been clarified. To clarify the mechanism of obesity-associated skin disorders, we aimed to characterize the skin function of subjects with obesity, and identify possible influencing factors. METHODS: Complex analyses including instrumental measurement, biochemical and lipidomics were performed for facial skin and physical evaluation in 93 Caucasian women with obesity (OB) and non-obesity (NOB). RESULTS: In OB, imbalance in metabolism of carbohydrate and lipid, autonomic nerve activity, and secreted factors were confirmed. In the skin properties in OB, surface roughness was higher by 70%, the water content was lower by 12%, and changes in the lipid profile of stratum corneum ceramide were observed; in particular, a 7% reduction of [NP]-type ceramide, compared with NOB. Moreover, significant redness accompanied by a 34% increase in skin blood flow was observed in OB. Correlation analysis elucidated that the water content was strongly correlated with local skin indices, such as the ceramide composition, redness, blood flow, and TNFα in the stratum corneum, whereas roughness was correlated with the systemic indices, such as serum insulin, leptin, and IL-6. CONCLUSIONS: Characteristics of obesity-associated skin were (A) reduction of the barrier and moisturizing function accompanied by intercellular lipid imbalance, (B) increased redness accompanied by hemodynamic changes, and (C) surface roughness. It was suggested that each symptom is due to different causes in local and/or systemic physiological impairment related to the autonomic nerve-vascular system, inflammation and insulin resistance.

Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine.

The purpose of the present study was to characterize rat organic anion transporter (Oat) 3 (Oat3, Slc22a8) in the efflux transport at the inner blood-retinal barrier (BRB). Reverse transcription-polymerase chain reaction analysis showed that rat (r) Oat3 mRNA is expressed in retinal vascular endothelial cells (RVECs), but not rOat1 and rOat2 mRNA. The expression of Oat3 in the retina and human cultured retinal endothelial cells was further confirmed by Western blot analysis. Immunohistochemical staining in RVECs showed that rOat3 is colocalized with glucose transporter 1, but not P-glycoprotein, suggesting that rOat3 is possibly located at the abluminal membrane of the RVEC. The contribution of rOat3 to the efflux of [(3)H]p-aminohippuric acid ([(3)H]PAH), [(3)H]benzylpenicillin ([(3)H]PCG), and [(14)C]6-mercaptopurine ([(14)C]6-MP), substrates of rOat3, from the vitreous humor/retina to the circulating blood across the inner BRB was evaluated using the microdialysis method. [(3)H]PAH, [(3)H]PCG, [(14)C]6-MP, and [(14)C] or [(3)H]d-mannitol, a bulk flow marker, were biexponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]PAH, [(3)H]PCG, and [(14)C]6-MP during the terminal phase was approximately 2-fold greater than that of d-mannitol. This efflux transport was reduced in the retinal presence of probenecid, PAH, and PCG, whereas it was not inhibited by digoxin. In conclusion, rOat3 is expressed at the inner BRB and involved in the vitreous humor/retina-to-blood transport of PAH, PCG, and 6-MP. This transport system is one mechanism to limit the retinal distribution of PAH, PCG, and 6-MP.

Facial application of high-concentration carbon dioxide prevents epidermal impairment associated with environmental changes.

PURPOSE: The transdermal application of carbon dioxide (CO2) gas dissolved in a solution and bathing in carbonated springs have been known to improve circulatory disorders. We aimed to elucidate and profile the effects of CO2 application on local skin function. PATIENTS AND METHODS: A liquid formulation that included high-concentration CO2 or a control formulation was applied to the face of healthy men for 8 weeks. Quantitative analysis was performed during the dry winter months. RESULTS: At the site where the control formulation was applied, transepidermal water loss (TEWL) increased while the moisturizing function (conductance) of facial skin decreased during the study period. However, at the CO2-treated site, increases in TEWL and decreases in conductance were significantly suppressed. In addition, the deterioration in scaliness and wrinkles parameters were suppressed by ≥40% at the CO2-treated site. There were no significant differences in skin surface pH or color properties between the control and test sites. CONCLUSION: This study suggests that the continuous application of a high-concentration CO2 formulation can affect skin physiology and has the potential to suppress reductions in the barrier and moisturizing functions of the stratum corneum accompanied by desquamation, which occurs during the winter.

Rapid desensitization of lipolysis in the visceral and subcutaneous adipocytes of rats.

In adipocytes, short and long term stimulation of beta adrenergic receptors (beta AR) induces the desensitization to catecholamines, leading to a decrease in the intracellular accumulation of cAMP, but the roles played by this in lipolysis is not clear. In this study, we assessed the catecholamine-induced desensitization of lipolysis and compared this in adipocytes isolated from visceral and subcutaneous fat tissues of rats. When adipocytes were pretreated with isoproterenol (ISO), the norepinephrine (NE)-induced lipolysis was significantly reduced dose- and time-dependently. A similar reduction of the lipolytic response was also found in NE-, dobutamine-, terbutaline- or BRL37344-induced lipolysis. The ISO- and each beta AR agonist-induced lipolysis in the visceral fat was not only higher than in the subcutaneous fat, but also markedly reduced by ISO- or NE-pretreatment. These results showed that short-term treatment of three subtypes of beta AR by each agonist induces a rapid reduction in the lipolytic response to beta AR stimulation. This suggests some common mechanism for the rapid desensitization of beta AR-agonist-induced lipolysis, in contrast with previous reports on the characteristics of beta AR subtypes. In addition, the regional difference of adipose tissue not only in inducing lipolysis but also in rapid desensitization was also apparent.

Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells.

The mechanism that removes homovanillic acid (HVA), an end metabolite of dopamine, from the brain is still poorly understood. The purpose of this study is to identify and characterize the brain-to-blood HVA efflux transporter at the rat blood-brain barrier (BBB). Using the Brain Efflux Index method, the apparent in vivo efflux rate constant of [3H]HVA from the brain, k(eff), was determined to be 1.69 x 10(-2) minute(-1). This elimination was significantly inhibited by para-aminohippuric acid (PAH), benzylpenicillin, indoxyl sulfate, and cimetidine, suggesting the involvement of rat organic anion transporter 3 (rOAT3). rOAT3-expressing oocytes exhibited [3H]HVA uptake (K(m) = 274 micromol/L), which was inhibited by several organic anions, such as PAH, indoxyl sulfate, octanoic acid, and metabolites of monoamine neurotransmitters. Neurotransmitters themselves did not affect the uptake. Furthermore, immunohistochemical analysis suggested that rOAT3 is localized at the abluminal membrane of brain capillary endothelial cells. These results provide the first evidence that rOAT3 is expressed at the abluminal membrane of the rat BBB and is involved in the brain-to-blood transport of HVA. This HVA efflux transport system is likely to play an important role in controlling the level of HVA in the CNS.

Characteristic expression of extracellular matrix in subcutaneous adipose tissue development and adipogenesis; comparison with visceral adipose tissue.

Adipose tissue is a connective tissue specified for energy metabolism and endocrines, but functional differences between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) have not been fully elucidated. To reveal the physiological role of SAT, we characterized in vivo tissue development and in vitro adipocyte differentiation. In a DNA microarray analysis of SAT and VAT in Wistar rats, functional annotation clusters of extracellular matrix (ECM)-related genes were found in SAT, and major ECM molecules expressed in adipose tissues were profiled. In a histological analysis and quantitative expression analysis, ECM expression patterns could be classified into two types: (i) a histogenesis-correlated type such as type IV and XV collagen, and laminin subunits, (ii) a high-SAT expression type such as type I, III, and V collagen and minor characteristic collagens. Type (i) was related to basal membrane and up-regulated in differentiated 3T3-L1 cells and in histogenesis at depot-specific timings. In contrast, type (ii) was related to fibrous forming and highly expressed in 3T3-L1 preadipocytes. Exceptionally, fibronectin was abundant in developed adipose tissue, although it was highly expressed in 3T3-L1 preadipocytes. The present study showed that adipose tissues site-specifically regulate molecular type and timing of ECM expression, and suggests that these characteristic ECM molecules provide a critical microenvironment, which may affect bioactivity of adipocyte itself and interacts with other tissues. It must be important to consider the depot-specific property for the treatment of obesity-related disorders, dermal dysfunction and for the tissue regeneration.

Body fat mass reduction and up-regulation of uncoupling protein by novel lipolysis-promoting plant extract.

We have found natural products exhibiting lipolysis-promoting activity in subcutaneous adipocytes, which are less sensitive to hormones than visceral adipocytes. The activities and a action mechanisms of a novel plant extract of Cirsium oligophyllum (CE) were investigated in isolated adipocytes from rat subcutaneous fat, and its fat-reducing effects by peroral administration and topical application were evaluated in vivo. CE-induced lipolysis was synergistically enhanced by caffeine, a phosphodiesterase inhibitor, and was reduced by propranolol, a beta adrenergic antagonist. The peroral administration of 10% CE solution to Wistar rats for 32 days reduced body weight gain, subcutaneous, and visceral fat weights by 6.6, 26.2, and 3.0%, respectively, as compared to the control group. By the topical application of 2% of this extract to rats for 7 days, weight of subcutaneous fat in the treated skin was reduced by 23.2%. This fat mass reduction was accompanied by the up-regulation of uncoupling protein 1 (UCP), a principal thermogenic mitochondrial molecule related to energy dissipating, in subcutaneous fat and UCP3 in skin except for the fat layer. These results indicate that CE promotes lipolysis via a mechanism involving the beta adrenergic receptor, and affects the body fat mass. This fat reduction may be partially due to UCP up-regulation in the skin including subcutaneous fat. This is the first report showing that repeated lipolysis promotion through CE administration may be beneficial for the systematic suppression of body fat accumulation or the control of fat distribution in obesity.

Enhancement of lipolytic responsiveness of adipocytes by novel plant extract in rat.

Subcutaneous adipocytes accumulate excess energy as triglycerides, but lipolytic response is less sensitive to catecholamines than visceral adipocytes. Obesity also induces catecholamine resistance of adipocytes. We have searched for crude drugs that could enhance the lipolytic response to noradrenalin. In this study, the lipolysis-promoting activities and action mechanisms of a novel plant extract from Hemerocallis fulva (HE) were investigated in isolated adipocytes from rat subcutaneous fat. HE exhibited no lipolysis-promoting activity alone but markedly promoted lipolysis when combined with noradrenaline; however, this synergistic activity was accompanied by no increase of intracellular cAMP production. This activity of HE was also observed when combined with cAMP analogue and was further enhanced by phosphodiesterase inhibitor. PKA inhibitor could reduce these activities of HE. These results indicate that HE is a novel lipolysis-promoting material that can sensitize the lipolytic response of adipocytes to catecholamine and suggest that HE can amplify the intra-cellular signaling pathway related to PKA or modify the other mechanism-regulating lipase activity. This characteristic material could contribute to improvement of adipose mobility in obesity-related disorder or in subcutaneous adiposity and to suppression of body fat accumulation.

Expression of uncoupling proteins in human skin and skin-derived cells.

Uncoupling protein (UCP) is a mitochondrial membrane protein that uncouples oxidative phosphorylation. The physiological function of major isoforms of UCPs is related to the control of body temperature and reactive oxygen species production. Although skin is an important organ for heat radiation and protection against stress, the expression and function of UCPs in the skin have remained unclear. The expression of UCPs in human skin and its derived cells was researched at the mRNA and protein levels. The effects of norepinephrine (NE) and 9-cis retinoic acid (RA) on UCP expression were also investigated. The expression of UCP1 mRNA was found in the human epidermis and was upregulated in differentiated keratinocytes. UCP1 expression in keratinocytes was synergistically upregulated by NE and RA treatment. Significant expression of UCP2 and UCP3 was observed also in cultured keratinocytes and fibroblasts. By immunohistochemistry, localization of UCP1 was found in the granular layer of the epidermis, sweat glands, hair follicles, and sebaceous glands of various sites in the human body. UCP3 was widely found in the dermis. This showed that UCPs exist in human skin, with their expression being under hormonal control. These findings are in stark contrast with the well-accepted view of UCP1 expression being exclusive to brown adipose tissue.

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells.

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.

Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers.

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3'-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR.

Mouse reduced in osteosclerosis transporter functions as an organic anion transporter 3 and is localized at abluminal membrane of blood-brain barrier.

The "reduced in osteosclerosis" transporter (Roct), which shows decreased expression in the osteosclerosis (oc) mutant mouse, has high homology with rat and human organic anion transporter 3 (OAT3). However, its transport properties and involvement in bone turnover are poorly understood. Here, we examined Roct-mediated transport using a Xenopus laevis oocyte expression system. Roct-expressing oocytes exhibited uptake of [(3)H]estrone sulfate, [(3)H]p-aminohippuric acid, [(3)H]benzylpenicillin, [(3)H]estradiol 17beta-glucronide, [(3)H]indoxyl sulfate, [(14)C]indomethacin, [(3)H]homovanillic acid, [(3)H]cimetidine, [(14)C]glutarate, [(14)C]salicylic acid, and [(3)H]methotrexate. Furthermore, the uptake of [(3)H]benzylpenicillin by Roct coexpressed with Na(+)-dicarboxylate cotransporter was trans-stimulated by glutarate preloading, and [(3)H]estrone sulfate uptake showed a similar tendency, suggesting that Roct is a dicarboxylate exchanger. [(3)H]Benzylpenicillin uptake by Roct was inhibited by OAT3 substrates and inhibitors, and by sulfate or glucuronide conjugates, and compounds involved in bone turnover. Roct mRNA is expressed abundantly in the kidney and was also detected in the brain, choroid plexus, and eye. Immunohistochemical analysis revealed that Roct is localized in brain capillary endothelial cells. These results indicate that the transport properties and tissue distribution of Roct are similar to those of OAT3, suggesting that Roct functions as mouse OAT3. Because Roct is expressed in the kidney and at the blood-brain barrier, it may play a role in the excretion of substrates such as conjugates and bone turnover factors.

Major role of organic anion transporter 3 in the transport of indoxyl sulfate in the kidney.

BACKGROUND: Indoxyl sulfate is a uremic toxin that accumulates in the body because of the patient's inability to excrete it and it induces a number of uremic symptoms and leads to chronic renal failure. The functional failure of the excretion system for indoxyl sulfate causes its accumulation in blood. The purpose of the present study was to characterize the transport mechanism responsible for the renal excretion of indoxyl sulfate. METHODS: The [3H]indoxyl sulfate transport mechanism was investigated using an in vivo tissue-sampling single-injection technique, the kidney uptake index (KUI) method. Rat organic anion transporter 3 (rOAT3)-expressing Xenopus laevis oocyte system was used for measuring [3H]indoxyl sulfate uptake activity. RESULTS: Probenecid showed a concentration-dependent inhibitory effect on the uptake of [3H]indoxyl sulfate using the KUI method, and uptake was inhibited by organic anions such as para-aminohippuric acid (PAH) and benzylpenicillin, by weak base such as cimetidine, and by uremic toxins, such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) and hippuric acid (HA). However, salicylic acid, indomethacin, 3,5,3'-triiodo-l-thyronine and indole acetic acid (IA) had no effect on the uptake. rOAT3-expressing oocytes exhibited uptake of [3H]indoxyl sulfate by rOAT3 (Km = 158 micromol/L). Moreover, a number of uremic toxins inhibited the uptake of [3H]indoxyl sulfate by rOAT3. CONCLUSIONS: These results suggest that rOAT3 is responsible for the renal uptake of indoxyl sulfate, and uremic toxins share the transport mechanism for indoxyl sulfate. Mutual inhibition of these uremic toxins via OAT3 may accelerate their accumulation in the body and, thereby, the progression of nephrotoxicity in uremia.

Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs.

Thiopurines are used as antileukemic drugs. However, during chemotherapy CNS relapses occur due to the proliferation of leukemic cells in the CNS resulting from restricted drug distribution in the brain. The molecular mechanism for this limited cerebral distribution remains unclear. The purpose of this study was to identify the transporter responsible for the brain-to-blood transport of thiopurines across the blood-brain barrier (BBB) using the brain efflux index method. [14C]6-Mercaptopurine (6-MP) and [3H]6-thioguanine were eliminated from rat brain in a time-dependent manner. The elimination of [14C]6-MP was inhibited by substrates of rat organic anion transporters (rOATs), including indomethacin and benzylpenicillin. rOAT1 and rOAT3 exhibited 6-MP uptake, while benzylpenicillin inhibited rOAT3-mediated uptake, but not that by rOAT1. rOAT3-mediated [14C]6-MP uptake was also inhibited by other thiopurine derivatives. Although methotrexate inhibited rOAT3-mediated [14C]6-MP uptake, the Ki value was 17.5-fold greater than the estimated brain concentration of methotrexate in patients receiving chemotherapy. Accordingly, 6-MP would undergo efflux transport by OAT3 from the brain without any inhibitory effect from coadministered methotrexate in the chemotherapy. In conclusion, rOAT3 is involved in the brain-to-blood transport of thiopurines at the BBB and is one mechanism of limited cerebral distribution.