The PDE4 inhibitor CHF6001 affects keratinocyte proliferation via cellular redox pathways.

Psoriasis is a skin disease characterized by abnormal keratinocyte proliferation and inflammation. Currently, there are no cures for this disease, so the goal of treatment is to decrease inflammation and slow down the associated rapid cell growth and shedding. Recent advances have led to the usage of phosphodiesterase 4 (PDE4) inhibitors for treatment of this condition. For example, apremilast is an oral, selective PDE4 inhibitor that is able to reduce skin inflammation and is Food and Drug Administration (FDA)-approved to treat adults with moderate to severe psoriasis and/or psoriatic arthritis. However, common target-related adverse events, including diarrhea, nausea, headache, and insomnia limit the usage of this drug. To circumvent these effects, the usage of PDE4 inhibitors specifically designed for topical treatment, such as CHF6001, may combine local anti-inflammatory activity with limited systemic exposure, improving tolerability. In this study, we showed that CHF6001, currently undergoing clinical development for COPD, suppresses human keratinocyte proliferation as assessed via BrdU incorporation. We also observed decreased re-epithelialization in a scratch-wound model after CHF6001 treatment. At the molecular level, CHF6001 inhibited translocation of phosphorylated NF-κB subunit p65, promoting loss of nuclear cyclin D1 accumulation and an increase of cell cycle inhibitor p21. Furthermore, CHF6001 decreased oxidative stress, measured by assessing lipid peroxidation (4-HNE adduct formation), through the inactivation of the NADPH oxidase. These results suggest that CHF6001 has the potential to treat skin disorders associated with hyperproliferative keratinocytes, such as psoriasis by targeting oxidative stress, abnormal re-epithelization, and inflammation.

SR-B1 involvement in keratinocytes in vitro wound closure.

Skin represents the most extended organ of human body, having as main function the protection of our body from outdoor stressors. Its protective ability is compromised when the skin is disrupted as a consequence of mechanical insults. For this purpose, cutaneous tissue is equipped with an efficient and fine mechanism involved in repairing the wounded area. Among the numerous players that take part in the wound healing process, SR-B1 has been recently shown to have a role in keratinocyte re-epithelialization. SR-B1 is a mediator of cholesterol uptake from HDLs, whereas it is implicated in other cellular processes such as vitamins absorption, vesicle trafficking or pathogen identification. The aim of this study was to investigate the mechanisms involved in SR-B1 role in skin wound closure. Our in vitro data demonstrated that SR-B1 influenced keratinocyte proliferation and migration through a downregulation of nuclear cyclin D1 levels and active MMP9 expression respectively possibly in an NF-kB-dependent mechanism. In addition, SR-B1 was also able to modulate keratinocyte morphology into a pro-migratory cytoskeleton rearrangement. The present in vitro study suggests a new role of SRB1 as a possible new key player in cutaneous wound healing mechanism.

Impaired enzymatic defensive activity, mitochondrial dysfunction and proteasome activation are involved in RTT cell oxidative damage.

A strong correlation between oxidative stress (OS) and Rett syndrome (RTT), a rare neurodevelopmental disorder affecting females in the 95% of the cases, has been well documented although the source of OS and the effect of a redox imbalance in this pathology has not been yet investigated. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have demonstrated in RTT cells high levels of H2O2 and HNE protein adducts. These findings correlated with the constitutive activation of NADPH-oxidase (NOX) and that was prevented by a NOX inhibitor and iron chelator pre-treatment, showing its direct involvement. In parallel, we demonstrated an increase in mitochondrial oxidant production, altered mitochondrial biogenesis and impaired proteasome activity in RTT samples. Further, we found that the key cellular defensive enzymes: glutathione peroxidase, superoxide dismutase and thioredoxin reductases activities were also significantly lower in RTT. Taken all together, our findings suggest that the systemic OS levels in RTT can be a consequence of both: increased endogenous oxidants as well as altered mitochondrial biogenesis with a decreased activity of defensive enzymes that leads to posttranslational oxidant protein modification and a proteasome activity impairment.

Effect of new curcumin-containing nanostructured lipid dispersions on human keratinocytes proliferative responses.

This study describes the production and characterization of nanostructured lipid dispersions (NLDs) containing curcumin (CUR) as new tools for curcumin topical delivery. Four types of NLDs based on monoolein in association with different emulsifiers were produced: Na cholate and poloxamer 407 (NLD1), poloxamer alone (NLD2), the mixture of Na cholate and Na caseinate (NLD3) and Na cholate alone (NLD4). Morphology and dimensional distribution of lipid dispersions were investigated by cryo-TEM and photon correlation spectroscopy (PCS). In vitro studies based on Franz cell, membrane nylon and stratum corneum-epidermis (SCE) were carried out to compare the four NLDs in terms of cytotoxicity in human keratinocytes and CUR diffusion. Our PCS studies showed differences in particles diameter among the different NLDs. In addition, cytotoxicity results in HaCaT cells evidenced that NLD1 and NLD2 were toxic at doses over 1 µm. Therefore, cryo-TEM was determined only for NLD3 and NLD4 showing that CUR did not affect their structure. Diffusion measurement in SCE and nylon membrane evidenced that CUR had a time-delayed release for NLD4. The 'wound healing' effect of NLD3 and NLD4 with and without CUR analysed keratinocytes in vitro, and a clear inhibition of cell proliferation/migration by CUR was observed. This effect was mediated by the inhibition of cyclin D1 expression as a consequence of the impaired NFkB activation. This study confirms the antiproliferative properties of CUR and evidenced a new possible model of CUR topical delivery for hyperproliferative cutaneous diseases such as psoriasis.

Accumulation of central fat correlates with an adverse oxidative balance in non-obese postmenopausal women.

The aim of the present study was to investigate whether accumulation of central fat is correlated with systemic oxidative stress (OxS) in non-obese apparently healthy postmenopausal women. Serum parameters of OxS (hydroperoxides and non-enzymatic antioxidants) along with body fat distribution, as assessed by dual-energy-X-ray absorptiometry (DXA), were evaluated in 134 non-obese postmenopausal women. Multiple regression analysis showed that central (trunk) fat significantly correlated with both markers of OxS independently of confounding factors (i.e. BMI, smoking, age, hypertension, legs and arms fat mass). In specific, the standardized regression coefficient was positive for hydroperoxides (ß = 0.324, p < 0.05) and negative for antioxidants (ß = -0.495, p < 0.01) level. In conclusion, the current data showed that the increase in central fat is an independent predictor of OxS condition among non-obese women in postmenopausal status. The possible pro-oxidant effects of the excess in central adiposity might be more harmful among post- than among pre-menopausal women, due to the postulated ability of E2 to contrast oxidative challenge and the related diseases.

A plasma proteomic approach in Rett syndrome: classical versus preserved speech variant.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.

Nitric Oxide/Cyclic GMP-Dependent Calcium Signalling Mediates IL-6- and TNF-α-Induced Expression of Glial Fibrillary Acid Protein.

Astrocyte activation is characterized by hypertrophy with increased glial fibrillary acidic protein (GFAP), whose expression may involve pro-inflammatory cytokines. In this study, the effects of pro-inflammatory IL-6 and TNF-α and anti-inflammatory cytokines IL-4 and IL-10 on nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signalling, intracellular calcium concentration ([Ca2+]i) and GFAP expression were investigated. In human glioblastoma astrocytoma U-373 MG cells, IL-6 and TNF-α, but not IL-4 or IL-10, increased iNOS, cGMP, [Ca2+]i and GFAP expression. The inhibitors of iNOS (1400 W), soluble guanylyl cyclase (ODQ) and IP3 receptors (ryanodine and 2-APB) reversed the increase in cGMP or [Ca2+]i, respectively, and prevented GFAP expression. In rat striatal slices, IL-6 and TNF-α, at variance with IL-4 and IL-10, promoted a concentration-dependent increase in Ca2+ efflux, an effect prevented by 1400 W, ODQ and RY/2APB. These data were confirmed by in vivo studies, where IL-6, TNF-α or the NO donor DETA/NO injected in the striatum of anaesthetised rats increased cGMP levels and increased GFAP expression. The present findings point to NO/cGMP-dependent calcium signalling as part of the mechanism mediating IL-6- and TNF-α-induced GFAP expression. As this process plays a fundamental role in driving neurotoxicity, targeting NO/cGMP-dependent calcium signalling may constitute a new approach for therapeutic interventions in neurological disorders.

The effects of hypoxia/reoxygenation on the physiological behaviour of U373-MG astrocytes.

Nerve cells are very susceptible to hypoxia responsive for mitochondrial dysfunctions involved in the subsequent oxidative stress, apoptosis and necrosis. In this paper, we examined the effect of 12 h incubation of U-373 MG astrocytes in hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell proliferation, modifications of NO and ATP production, intracellular Ca(2+) concentration [Ca(2+)](i), membrane potential, desferoxamine-chelatable free iron, esterified F2-isoprostanes levels and the production of phosphorylated ERK. The same parameters were evaluated also after a following re-oxygenation period of 24 h. Immediately after hypoxia the NO concentration increased significantly and returned to values similar to those of controls after the re-oxygenation period. At the same time, ATP levels remained similar to controls and the cell proliferation significantly decreased. This involved a significant increase of [Ca(2+)](i) immediately after hypoxia and the value remained significantly elevated after the following re-oxygenation period. Moreover, after hypoxia, astrocytes were slightly although not significantly depolarized. Indeed iron and F2-isoprostanes levels increased significantly after hypoxia. Finally ERK proteins increased slowly and not significantly after hypoxia and the same trend was observed after the re-oxygenation period. On the whole, our results indicate that 2% O(2) hypoxia induces a moderate oxidative stress, well tolerated by U-373 MG cells, remaining the ATP production, mitochondrial membrane potential and activated ERK proteins, similar to the values of controls.

The physiological behaviour of IMR-32 neuroblastoma cells is affected by a 12-h hypoxia/24-h reoxygenation period.

Nervous system cells are highly dependent on adequate tissue oxygenation and are very susceptible to hypoxia, which causes mitochondrial dysfunctions involved in apoptosis and necrosis. In this paper, we examine the effect of a 12-h incubation of differentiated IMR-32 neuroblastoma cells in a hypoxic environment (73% N(2): 2% O(2): 5% CO(2), v:v) by evaluating cell viability, modifications of NO, intracellular Ca(2+) concentration [Ca(2+)](i) and membrane potential, the production of phosphorylated ERK, desferoxamine-chelatable free iron and esterified F2-isoprostane levels. The same parameters were evaluated after a subsequent 24-h re-oxygenation period. The NO concentration increased significantly immediately after hypoxia and returned to values similar to those of controls after the reoxygenation period. At the same time, we observed a significant increase of [Ca(2+)](i) immediately after hypoxia. Phosphorylated ERK proteins increased significantly during the first 2 h of hypoxia, then decreased, and remained practically unmodified after 12 h hypoxia and the following reoxygenation period. Moreover, IMR-32 cell mitochondria were significantly depolarized after hypoxia, while membrane potential returned to normal after the reoxygenation period. Finally, desferoxamine-chelatable free iron and F2-isoprostane levels also increased significantly after hypoxia. Our results indicate that 2% O(2) hypoxia induces variations of NO and [Ca(2+)](i) with subsequent mitochondrial depolarization, and it is responsible for oxidative stress, represented by increased free iron and F2-isoprostane, protein carbonyls and 4 hydroxynonenal protein adducts levels.

Cigarette smoke affects ABCAl expression via liver X receptor nuclear translocation in human keratinocytes.

Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.

Correction: Cigarette Smoke Affects Keratinocytes SRB1 Expression and Localization via H2O2 Production and HNE Protein Adducts Formation.

[This corrects the article DOI: 10.1371/journal.pone.0033592.].

Modulation of cutaneous scavenger receptor B1 levels by exogenous stressors impairs "in vitro" wound closure.

Scavenger receptor B1 (SR-B1) is a trans-membrane protein, involved in tissue reverse cholesterol transport. Several studies have demonstrated that SR-B1 is also implicated in other physiological processes, such as bacteria and apoptotic cells recognition and regulation of intracellular tocopherol and carotenoids levels. Among the tissues where it is localized, SR-B1 has been shown to be significantly expressed in human epidermis. Our group has demonstrated that SR-B1 levels are down-regulated in human cultured keratinocytes by environmental stressors, such as cigarette smoke, via cellular redox imbalance. Our present study aimed to investigate whether such down-regulation was confirmed in a 3D skin model and under other environmental challengers such as particulate matter and ozone. We also investigated the association between oxidation-induced SR-B1 modulation and impaired wound closure. The data obtained showed that not only cigarette, but also the other environmental stressors reduced SR-B1 expression in epidermal cutaneous tissues and that this effect might be involved in impaired wound healing.

Tropospheric ozone affects SRB1 levels via oxidative post-translational modifications in lung cells.

Exposure to air pollution is associated with increased respiratory morbidities and susceptibility to lung dysfunction. Ozone (O3) is commonly recognized as one of the most noxious air pollutant and has been associated with several lung pathologies. It has been demonstrated that decreased lung disorder severity and incidence are connected with the consumption of a diet rich in fruits and vegetables, suggesting that higher intake of dietary micronutrients and phytoactive compounds can be beneficial. However, dietary supplementation - i.e. vitamin E (α-tocopherol) or vitamin A - has not always been effective in improving pulmonary function. Recently, research on the role of nutritional antioxidants on human health has focused more on studying their uptake at the cellular level rather than their effective ability to scavenge reactvive oxygen species (ROS). The Scavenger Receptor B1 (SRB1) has been shown to play a prominent role in the uptake, delivery and regulation of vitamin E in the lung. Given the importance of SRB1 in maintaining lung tissue in a healthy condition, we hypothesize that its expression could be modulated by pollution exposure, which thus could indirectly affect the uptake and/or delivery of lipophilic substances, such as vitamin E. To characterize the molecular mechanism involved in the redox modulation of SRB1, its cellular levels were assessed in human alveolar epithelial cells after O3 exposure. The results demonstrated that O3 induced the loss of SRB1 protein levels. This decline seems to be driven by hydrogen peroxide (H2O2) as a consequence of an increased activation of cellular NADPH oxidase (NOX), as demonstrated by the use of NOX inhibitors or catalase that reversed this effect. Furthermore, O3 caused the formation of SRB1-aldheyde adducts (4-hydroxy-2-nonenal) and the consequent increase of its ubiquitination, a mechanism that could account for SRB1 protein loss.

In vitro vascular toxicity of tariquidar, a potential tool for in vivo PET studies.

The P-glicoprotein (P-gp) inhibitor tariquidar is used to detect functional alterations of blood brain barrier pumps in PET imaging. The doses required, however, up to 4-fold higher than those already used in clinical trials to reverse multidrug resistance, cause syncopal episode and hypotension. Therefore, the effects of these doses toward the vasculature were investigated and an in-depth analysis of tariquidar-mediated effects on A7r5 and EA.hy926 cells viability, on the mechanical activity of freshly and cultured rat aorta rings and on L-type Ca2+ current [ICa(L)] of A7r5 cells has been performed. In both A7r5 and EA.hy926 cells, tariquidar was not cytotoxic up to 1µM concentration. On the contrary, at 10µM, it caused apoptosis already after 24h treatment. In fresh aorta rings, 10µM tariquidar partially relaxed phenylephrine-, but not 60mM K+ (K60)-induced contraction. In rings treated with 10µM tariquidar for 7days, the contractile response to both phenylephrine and K60 remained unchanged. Finally, tariquidar did not modify ICa1.2 intensity and kinetics. In conclusion, Tariquidar might exert both cytotoxic and acute, weak vascular effects at concentrations comparable to those employed in PET imaging. This implies that caution should be exercised when using it as diagnostic tool.

Resveratrol: from diet to topical usage.

The stilbene derivative resveratrol (3,5,4'-trihydroxy-stilbene; RESV) has become the subject of interest of many researchers and the pharmaceutical industries due to its well-acclaimed beneficial biological activities. Although earlier research tended to focus on the effects of RESV on cardiovascular disorders, many other studies have described the beneficial effects of RESV in the areas of cancer chemoprevention and inflammation and interest of researchers on this compound is still increasing. It is now well accepted that the effect of RESV is not just due to its so called "antioxidant" activity but mainly (if not only) because of the ability of this compound to trigger cell signaling pathways and gene expression involved in cellular defense systems. Many "in vitro" studies on RESV did not take into account that although its oral absorption is about 75% it undergoes rapid metabolism and the concentration in the blood stream is almost undetectable. For this reason interest in the topical usage of RESV by cosmeceutical skin care brands has exponentially increased in the last decade reporting in general very promising results on its beneficial effect in protecting the skin from outdoor insults, but there is still some controversy on its topical usage mainly surrounding the concentration used. Therefore, more basic research on the topical application of RESV should be performed to better understand the way it prevents cutaneous damage and whether it could be recommended as a preventive skin aging agent for all skin insults.

Vascular Toxicity Risk Assessment of MC18 and MC70, Novel Potential Diagnostic Tools for In Vivo PET Studies.

The P-glicoprotein (P-gp) inhibitor MC18 has been recently proposed as a valuable PET tracer to measure P-gp expression in vivo. The aim of this study was to evaluate the toxic hazard towards the vasculature of MC18 along with the structurally related and more potent P-gp inhibitor MC70. Their effects on A7r5 and EA.hy926 cells viability, on the mechanical activity of fresh and cultured rat aorta rings as well as on Cav 1.2 channel current (ICa1.2 ) of A7r5 cells were studied. At concentrations >10 µM, MC18 and MC70 decreased cell viability causing evident morphological changes. In fresh rat aorta rings, both compounds (0.1-100 µM) antagonized phenylephrine-induced contraction in a concentration-dependent manner, with IC50 values in the range of 1.67-14.49 µM, whereas only MC18 caused a concentration-dependent decrease of the 60 mM K+ (K60)-induced responses. In rings cultured for 7 days with both compounds (1-10 µM), 10 µM MC70 significantly reduced, while 10 µM MC18 completely prevented the contractile response to both phenylephrine and K60. MC18 and MC70 (0.1-100 µM) inhibited ICa1.2 in a concentration-dependent manner with IC50 values of 16.81 and 32.13 µM, respectively. These findings demonstrate that MC18-induced vascular effects took place at concentrations that are at least three orders of magnitude higher than those (≤10 nM) allowing in vivo measurement of P-gp expression. Thus, MC18, and possibly MC70, can be considered promising PET tools for in vivo P-gp quantification.

SRB1 as a new redox target of cigarette smoke in human sebocytes.

For its critical location, the skin represents the major interface between the body and the environment, therefore is one of the major biological barriers against the outdoor environmental stressors. Among the several oxidative environmental stressors, cigarette smoke (CS) has been associated with the development and worsening of many skin pathologies such as acne, dermatitis, delayed wound healing, aging and skin cancer. In our previous work we have demonstrated that CS is able to affect genes involved in skin cholesterol trafficking, among which SRB1, a receptor involved in the uptake of cholesterol from HDL, seems to be very susceptible to the oxidative stress induced by CS. In the present work we wanted to investigate the presence of SRB1 in human sebocytes and whether CS can affect cholesterol cellular uptake via the redox modulation of SRB1. By using a co-culture system of keratinocytes/sebocytes, we found that CS exposure induced a SRB1 protein loss without affecting sebocytes viability. The decrease of SRB1 levels was a consequence of SRB1/HNE adducts formation that leads to SRB1 ubiquitination and degradation. Moreover, the CS-induced loss of SRB1 induced an alteration of sebocytes lipid content, also demonstrated by cholesterol quantification in SRB1 siRNA experiments. In conclusion, exposure to CS, induced SRB1 post-translational modifications in sebocytes and this might affect sebocytes/skin functionality.

Ozone mediators effect on "in vitro" scratch wound closure.

The beneficial effect of low doses of ozone on wound healing has been well documented and attributed mainly to its bactericidal and pro-oxidant properties. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects are the results of oxidative mediators. Among the molecule produces by the interaction of ozone with biological systems, there are HNE and H2O2. At today, the cellular mechanisms accounting for the positive effects of mild ozonization on wound closure are still largely unexplored. The aim of the present study was to evaluate the effect of different non-toxic doses of ozonated saline ranging from 2 to 300 µM, in an in vitro wound scratch model by the use of human keratinocytes. The results showed that ozonated saline is able to improve in vitro wound healing by stimulating cell proliferation as measured by BrdU assay and PCNA protein levels. In order to better elucidate the molecules that play the main role in the beneficial effect of ozonated saline in wound healing, HNE and H2O2 were used alone or in combination to mimic ozonated saline effect. Surprisingly, keratinocytes treated with different doses of HNE and H2O2 did not significantly improve the wound closure, while the combination of the two compounds was able to improve wound closure. In addition, Nrf2 pathways were also activated as determined by its translocation to the nucleus and the increased HO1 gene expression. The present work suggests that ozonated saline effect on wound closure is the results of the combination of more molecules among which HNE and H2O2 play a key role.

Skin Damage Mechanisms Related to Airborne Particulate Matter Exposure.

Epidemiological studies suggest a correlation between increased airborne particulate matter (PM) and adverse health effects. The mechanisms of PM-health effects are believed to involve oxidative stress and inflammation. To evaluate the ability of PM promoting skin tissue damage, one of the main organs exposed to outdoor pollutants, we analyzed the effect of concentrated ambient particles (CAPs) in a reconstructed human epidermis (RHE) model. RHE tissues were exposed to 25 or 100 µg/ml CAPs for 24 or 48 h. Data showed that RHE seems to be more susceptible to CAPs-induced toxicity after 48 h exposure than after 24 h. We found a local reactive O(2) species (ROS) production increase generated from metals present on the particle, which contributes to lipids oxidation. Furthermore, as a consequence of altered redox status, NFkB nucleus translocation was increase upon CAPs exposure, as well as cyclooxygenase 2 and cytochrome P450 levels, which may be involved in the inflammatory response initiated by PM. CAPs also triggered an apoptotic process in skin. Surprisingly, by transition electron microscopy analysis we showed that CAPs were able to penetrate skin tissues. These findings contribute to the understanding of the cutaneous pathophysiological mechanisms initiated by CAPs exposure, where oxidative stress and inflammation may play predominant roles.