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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.

Correction for ‘Wound healing properties of hyaluronan derivatives bearing ferulate residues’ by Giuseppe Valacchi et al., J. Mater. Chem. B, 2015, DOI: 10.1039/c5tb00661a.

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.

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.

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.

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 O2 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. Fil: Magnani, Natalia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Muresan, Ximena M.. Universita Di Ferrara; Italia Fil: Belmonte, Giuseppe. Universita Di Ferrara; Italia Fil: Cervellati, Franco. Universita Di Ferrara; Italia Fil: Sticozzi, Claudia. Universita Di Ferrara; Italia Fil: Pecorelli, Alessandra. Universita Di Ferrara; Italia Fil: Miracco, Clelia. Università Degli Studi Di Siena; Italia Fil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Evelson, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Valacchi, Giuseppe. Universita Di Ferrara; Italia

Chronic obstructive pulmonary disease (COPD) has been recognized as one of the major causes of morbidity and mortality in the United States; it is the third leading cause of deaths in the United States, with approximately 15 million Americans affected with COPD. Although exposure to cigarette smoke has been shown to be the main, if not the only, risk factor for COPD, the mechanisms underlying this association remain unclear. Most smokers do not develop COPD, suggesting that a combination of exposure and susceptibility (genetic background) is required. Several mechanisms contribute to the pathogenesis of COPD, such as influx of inflammatory cells into the lung, imbalance between proteolytic and antiproteolytic molecules, disruption of the balance between apoptosis and replenishment of structural cells in the lung, and disruption of oxidant/antioxidant balance. The scavenger receptor BI (SRB1) plays an important role in mediating the uptake of high-density lipoprotein (HDL)-derived cholesterol and cholesteryl ester in tissues. In addition to its role as the HDL receptor, SRB1 is also involved in pathogen recognition, identification of apoptotic cells, tissue antioxidant uptake (tocopherol and carotenoids), and lung surfactant composition, all factors involved in COPD pathogenesis. Therefore, it is possible that lung SRB1 levels are involved in the development of COPD.

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. Fil: Muresan, Ximena Maria. Università di Ferrara; Italia Fil: Sticozzi, Claudia. Università di Ferrara; Italia Fil: Belmonte, Giuseppe. Università di Ferrara; Italia Fil: Savelli, Vinno. Università degli Studi di Siena; Italia Fil: Evelson, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Valacchi, Giuseppe. Università di Ferrara; Italia. North Carolina State University; Estados Unidos

Ethnopharmacological relevance Mucuna pruriens ( Mp ) is a plant belonging to the Fabaceae family, with several medicinal properties among which its potential to treat diseases where reactive oxygen species (ROS) play an important role in the pathogeneses. The aim was to investigate the effects of Mp leaf methanolic extract (MPME) on human keratinocytes protein expression and its role in preventing proteins oxidation after oxidative stress (OS) exposure. Material and methods The effects of MPME on HaCaT cells protein expression were evaluated treating cells with different concentrations of MPME, with glucose oxidase (GO, source of OS) and with MPME subsequently treated with GO. The protein patterns of treated HaCaT cells are analyzed by two-dimensional gel electrophoresis (2-DE) and compared with that of untreated HaCaT. Immunoblotting was then used to evaluate the role of MPME in preventing the 4-hydroxynonenal protein adducts (4-HNE PAs) formation (marker of OS). Results Eighteen proteins, identified by mass spectrometry (LC-ESI-CID-MS/MS), were modulated distinctly by MPME in HaCaT. Overall, MPME counteract GO effect, reducing the GO-induced overexpression of several proteins involved in stress response (T-complex protein 1, Protein disulfide-isomerase A3, Protein DJ-1, and Stress-induced-phosphoprotein 1), in cell energy methabolism (Inorganic pyrophosphatase, Triosephosphate isomerase isoform 1, 2-phosphopyruvate-hydratase alpha-enolase, and Fructose-bisphosphate aldolase A isoform 1), in cytoskeletal organization (Cytokeratins 18, 9, 2, Cofilin-1, Annexin A2 and F-actin-capping protein subunit beta isoform 1) and in cell cycle progression (Eukaryotic translation initiation factor 5A-1 isoform B). In addition, MPME decreased the 4-HNE PAs levels, in particular on 2-phosphopyruvate-hydratase alpha-enolase and Cytokeratin 9. Conclusions Our findings show that MPME might be helpful in the treatment of OS-related skin diseases by preventing protein post-translational modifications (4-HNE PAs).

Reactive astrogliosis, a feature of neuro-inflammation is induced by a number of endogenous mediators including cytokines. Despite interleukin-1 beta (IL-1β) stands out as the major inducer of this process, the underlying mechanism and its role on neuronal viability remain elusive. We investigated in human astrocytoma cells and the rat brain striatum, the role of the nuclear factor-kB (NF-kB) intracellular Ca 2+ concentration ([Ca 2+ ] i ) calmodulin (CaM) and extracellular regulated mitogen-activated protein kinases (ERK1/2) in IL-1β-induced expression of glial fibrillary acidic protein (GFAP) and neuronal apoptosis associated to a brain trauma. Cell data showed that IL-1β (1 ng/ml) increased NF-kB, pERK1/2 and GFAP expression. Nevertheless, further increase in IL-1β levels reversed progressively these responses. Preventing ERK1/2 activation with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]-butadiene antagonized IL-1β-induced GFAP expression while inhibiting selectively nuclear translocation of NF-kB with caffeic-acid phenethyl-ester down-regulated both ERK1/2 and GFAP expression induced by IL-1β. The GFAP response was also prevented by antagonizing selectively increase in [Ca 2+ ] i , CaM activity or inducible nitric oxide synthase expression with respectively ryanodine plus 2-aminoethoxydiphenyl-borate, N -(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride and N -[(3-(aminomethyl)-phenyl]methyl]-ethanimidamide dihydrochloride. Data in vivo supported these findings and showed that GFAP expression induced by IL-1β (50 ng/ml) correlated with attenuated glial scar formation and reduced neuronal apoptosis. Our data identified the NF-kB/Ca 2+ –CaM/ERK signaling pathway as a novel in vivo key regulator of IL-1β-induced astrogliosis which may represent a potential target in neurodegeneration.

Selective COX-2 inhibitors (COXib) belonging to the class of diaryl heterocycles (e.g., celecoxib, rofecoxib, etc.), are devoid of the undesirable effects due to their capacity to inhibit selectively inducible (COX-2), responsible for inflammatory effects but not constitutive cyclooxygenase-1 (COX-1)(COX); responsible for cytoprotective effects on gastric mucosa. In addition, several reports have identified an increased risk of cardiovascular events associated with the use of COXib. We have developed a new series of anti-inflammatory agents (1,5-diarylpyrrole-3-alkoxyethyl esters and ethers). To evaluate the effect of two 1,5-diarylpyrrole-3-alkoxyethyl ethers, VA441 and VA428 (up to 100 μM), respectively, in comparison with two well known COXib, celecoxib and rofecoxib, on HaCaT cell (keratinocytes) proliferation and toxicity. Crucial molecules in cell cycle progression, i.e. NFκB and ERK as targets/mediators and cyclin D1 and p21 Cip1/Kip as final effectors were evaluated by Western blot, immunohystochemistry and q-PCR analysis. Both compounds, VA441 and VA428, showed a strong inhibition of cell proliferation, and did not exhibit cytotoxicity. The anti-proliferative effect was accompanied by a strong activation of ERK and induction of the cell cycle inhibitor p21. In addition, there was a clear inhibition of the transcription factor NF-κB and downregulation of cyclin D1, with enforced inhibition of the HaCaT cell cycle progression. These data suggest that compounds VA441 and VA428, along with their role in inhibiting COX-2 and inflammation, could have a possible therapeutic (topical and systemic) use against skin proliferative disorders, such as psoriasis.

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.

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 Cav1.2 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. This article is protected by copyright. All rights reserved.

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.

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.

4-(Thiophen-2-yl)butanoic acid was identified as a cyclic substitute of the unsaturated alkyl chain of the natural ligand, capsaicin. Accordingly, a new class of amides was synthesized in good yield and high purity and their molecular recognition against the target was investigated by means of docking experiments followed by molecular dynamics simulations, in order to rationalize their geometrical and thermodynamic profiles. The pharmacological properties of these new compounds were expressed as activation (EC50) and desensitization (IC50) potencies. Several compounds were found to activate TRPV1 channels, and in particular, derivatives 1 and 10 behaved as TRPV1 agonists endowed with good efficacy as compared to capsaicin. The most promising compound 1 was also evaluated for its protective role against oxidative stress on keratinocytes and differentiated human neuroblastoma cell lines expressing the TRPV1 receptor as well as for its cytotoxicity and analgesic activity in vivo.

Mucuna pruriens (Fabaceae) is an established herbal drug used for the management of male infertility, nervous disorders, and also as an aphrodisiac. It has been shown that its seeds are potentially of substantial medicinal importance. The ancient Indian medical system, Ayurveda, traditionally used M. pruriens, even to treat such things as Parkinson's disease. M. pruriens has been shown to have anti-parkinson and neuroprotective effects, which may be related to its anti-oxidant activity. In addition, anti-oxidant activity of M. pruriens has been also demonstrated in vitro by its ability to scavenge DPPH radicals and reactive oxygen species. In this review the medicinal properties of M. pruriens are summarized, taking in consideration the studies that have used the seeds extracts and the leaves extracts.

Living organisms are continuously exposed to environmental pollutants. Because of its critical location, the skin is a major interface between the body and the environment and provides a biological barrier against an array of chemical and physical environmental pollutants. The skin can be defined as our first defense against the environment because of its constant exposure to oxidants, including ultraviolet (UV) radiation and other environmental pollutants such as diesel fuel exhaust, cigarette smoke (CS), halogenated hydrocarbons, heavy metals, and ozone (O₃). The exposure to environmental pro-oxidant agents leads to the formation of reactive oxygen species (ROS) and the generation of bioactive molecules that can damage skin cells. This short review provides an overview of the effects and mechanisms of action of CS, O₃, and UV on cutanous tissues.

The intervention strategies in various types of skin wounds include several treatment programs that depend on the identified disease. Several factors such as aging, defective nutrition, traumatism, atherosclerosis, and diabetes may contribute to the formation of a wound that has no tendency to heal due to a defective and complicated repair process. The numerous advances in the understanding of the wound-healing process in both acute and chronic lesions have been recently described. The purpose of this paper is to describe relatively new approaches as viable alternatives to current wound-healing therapies. The future challenges for both the best targeting and optimization of these potential treatments are also described.

The scavenger receptor class B type I (SR-B1) plays an important role in meditating the uptake of HDL-derived cholesterol and cholesteryl ester in the liver and steroidogenic tissues. In addition to being ubiquitous, SR-B1 is a high-density lipoprotein (HDL) receptor in many tissues, though the mechanism by which SR-B1 does this is unclear. Other than its role as an HDL receptor, SR-B1 is also involved in pathogen recognition; its expression can be modulated by lipopolysaccharide and oxidative stress; and it plays a significant role in the uptake of lipid soluble vitamins, such as vitamin E and carotenoids. In this short review, we have summarized the biological aspects to which SR-B1 has been thus far associated.

Scavenger receptor B1 (SRB1), is a membrane receptor involved in several physiological functions including cholesterol and lipid soluble vitamins uptake, pathogens recognition and vesicles trafficking. Although this receptor is mainly localized in the liver and steroidogenic tissues, it has been shown to be expressed also in human skin. Recent studies have evidenced SRB1 susceptibility to oxidative stress and considering that cutaneous tissue is the main organ exposed to the outdoor stressors, it was our interest to understand whether the exposure of skin to environmental pollutants could affect its expression and consequently skin physiology. To answer our questions, we have performed experiments with homemade 3D skin models exposed to different pollutants: ozone, PM and cigarette smoke. Surprisingly all the stressors were able to down-regulate SRB1. In addition, the loss of SRB1 (after the challenges or by siRNA) prevented tissue wound closure and proliferation. Moreover, by the use of SR-B1 knockdown organotypic skin model, we showed a striking change in lipids organization within the epidermis. Our findings suggest that SR-B1 loss upon outdoor insults may influence cutaneous repair and lipid homeostasis making it as a key player in maintaining skin homeostasis.

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.

Background and purpose: Evidence is accumulating to support a role for interleukin-1β (IL-1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated. Experimental approach: In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca2+ concentration ([Ca2+]i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation. Key results: Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca2+ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca2+]i. Conclusions and implications: These data identified the NO/Ca2+/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders. British Journal of Pharmacology (2008) 153, 1706–1717; doi:10.1038/bjp.2008.40; published online 25 February 2008

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births. A clear etiological factor present in more than 90% of classical RTT cases is the mutation of the gene encoding methyl-CpG-binding protein 2 (MECP2). Recent work from our group was able to shown a systemic oxidative stress (OxS) in these patients that correlates with the gravity of the clinical features. Using freshly isolated skin fibroblasts from RTT patients and healthy subjects, we have performed a two-dimensional gel electrophoresis in order to evidence the oxidative modifications of proteins with special focus on the formation of protein adducts with 4-hydroxynonenal (4-HNE PAs)-a major secondary product of lipid peroxidation- and Nitrotyrosine, a marker derived from the biochemical interaction of nitric oxide (NO) or nitric oxide-derived secondary products with reactive oxygen species (ROS). Then, oxidatively modified spots were identified by mass spectrometry, LC-ESI-CID-MS/MS. Our results showed that 15 protein spots presented 4-HNE PAs and/or nitrotyrosine adducts in fibroblasts proteome from RTT patients compared to healthy control cells. Post-translationally modified proteins were related to several functional categories, in particular to cytoskeleton structure and protein folding. In addition, clear upregulated expression of the inducible NO synthase (iNOS) with high nitrite levels were observed in RTT fibroblasts, justifying the increased nitrotyrosine protein modifications. The present work describes not only the proteomic profile in RTT fibroblasts, but also identifies the modified proteins by 4-HNE and nitrotyrosine. Of note, for the first time, it appears that a dysregulation of NO pathway can be associated to RTT pathophysiology. In conclusion, the evidence of a wide range of proteins able to forms adducts with 4-HNE, Nitrotyrosine or with both confirms the possible alteration of several aspects of cellular functions that well correlates to the complex clinical features of RTT patients.

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers were previously synthesized and the potential anti-inflammatory and antinociceptive activities of these compounds were evaluated in vivo. The compounds displayed a very good activity against both carrageenan-induced hyperalgesia and oedema in the rat paw test. Therefore, in a very preliminary test, compounds (8a,b) showed antiproliferative activity in the HaCaT (aneuploid immortal keratinocyte from adult human skin) cell models. On these basis, our research continued with the synthesis of fluorinated derivatives (8c,d, 9b-d, and 10b-d) with the aim of improving the pharmacokinetic profile of the previous active compounds. Substitution of a hydrogen atom by a fluorine atom may change the conformational preferences of the molecules due to stereoelectronic effects and also fluorine atom may be able to exert the metabolic obstruction reducing the “first-pass effect”. Compound 10b exhibited a prominent in vivo anti-inflammatory and antinociceptive activities, in addition its antiproliferative power in an in vitro model of human skin cancer is herein described.

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. (C) 2015 Elsevier B.V. All rights reserved.

Microtubules are composed by α- and β-tubulin polypeptides. α-tubulin undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyrtubulin). Recent studies have shown that α-tubulin tyrosine residues can be nitrated and the incorporation of NO2Tyr into the C-terminus of Glu-tubulin forms a complex that blocks the tyrosination/detyrosination cycle, an event that can compromise protein/enzyme functions, such as cell division. Since many studies demonstrated that Glu-tubulin levels increase in cancer, the aim of the present study was to investigate the effect of new drugs, fluorazone derivatives (K1-K2-K9-K10-K11), on the proliferation of melanoma cells. Our results demonstrated that these drugs, except for K2, were able to inhibit cellular proliferation without exhibiting cytotoxicity. The anti-proliferative effect was accompanied by the decrease of Glu-tubulin levels and the increase of its nitration. This effect seems to be a consequence of NO2 induction and NO2Tyr ligation to Glu-tubulin. Collectively, these results, showing that the fluorazone derivatives, by promoting NO2Tyr incorporation into α-tubulin, are able to arrest the cycle of detyrosination/tyrosination and to inhibit cell proliferation, offer new perspectives for the possible usage of these drugs, alone or in combination, as non-toxic, anti-proliferative agents in melanoma.

VA694, a promising cyclooxigenase-2 (COX-2)-inhibiting hybrid drug endowed with nitric oxide (NO) releasing properties (NO-COXIB), showed COX-2-selective inhibitory effects, associated with interesting anti-inflammatory and anti-nociceptive activities. Therefore, we studied the effects of VA694 on cartilage metabolism, in comparison with Naproxcinod, a COX inhibitor and NO donor (CINOD), and Naproxen, a traditional non-steroidal-anti-inflammatory drug (NSAID) on human osteoarthritic chondrocyte cultures. IL-1β-stimulated chondrocytes showed a significant decrease in cell viability (P

CDKL5 mutation is associated with an atypical Rett syndrome (RTT) variant. Recently, cholesterol homeostasis perturbation and oxidative-mediated loss of the high-density lipoprotein receptor SRB1 in typical RTT have been suggested. Here, we demonstrate an altered lipid serum profile also in CDKL5 patients with decreased levels of SRB1 and impaired activation of the defensive system Nrf2. In addition, CDKL5 fibroblasts showed an increase in 4-hydroxy-2-nonenal– and nitrotyrosine–SRB1 adducts that lead to its ubi-quitination and probable degradation. This study highlights a possible common denominator between two different RTT variants (MECP2 and CDKL5) and a possible common future therapeutic target.

Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balance through free radicals production or through the modulation of antioxidant enzymes. The respiratory tract is one of the organs directly exposed to CS and it is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeability. In this study, we have used a human lung epithelial cell line, Calu-3, to evaluate the effect of CS on lung epithelial cell junctions levels, with special focus on the expression of two proteins involved in intercellular communication: connexins (Cx) 40 and 43. CS exposure increased Cx40 gene expression but not of Cx43. CS also induced NFκB activation and the formation of 4HNE-Cxs adducts. Since corilagin, a natural polyphenol, is able to inhibit NFκB activation, we have determined whether corilagin could counteract the effect of CS on Cxs expression. Corilagin was able to diminish CS induced Cx40 gene expression, 4HNE-Cx40 adducts formation, and NFκB activation. The results of this study demonstrated that CS induced the loss of cellular junctions in lung epithelium, possibly as a consequence of Cx-4HNE adducts formation, and corilagin seems to be able to abolish these CS induced alterations.

Pollution is not a recent phenomenon. Air pollution has been a problem since the appearance of our earliest ancestors. But over the past few centuries, the rising human activities associated with population growth and industrialization have negatively contributed to the air quality.

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1∶10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: −1.82±0.15, −2.15±0.06, and −2.59±0.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.

The plant polyphenol, resveratrol (Resv, 3,4,5-trihydroxystilbene), naturally occurring in a number of fruits and other food products, has been extensively studied over the last two decades for its beneficial properties. Recently, its possible topical use in ameliorating skin conditions has also been proposed; however, its role in preventing cigarette smoke (CS)-induced keratinocyte damage has not been investigated yet. Because of its peculiar location, cutaneous tissue is constantly exposed to several environmental stressors, such as CS. Many compounds presented in CS, have been shown to induce, directly or indirectly, cellular oxidative stress (OS) and inflammation via the production of ROS and lipid peroxidation compounds, among which 4HNE has been shown to be one of the most reactive. In this study, we have shown that resveratrol (at a dose of 10 μM) can decrease CS-induced ROS and carbonyl formation in human keratinocytes. In addition, pre-treatment with resveratrol prevented the induction of TRPA1 expression (mRNA and protein levels), a known receptor involved in cellular differentiation and inflammation, which has been recently shown to be activated by 4HNE. Finally, in keratinocytes, resveratrol could increase the expression of MsrA, enzyme involved in cell defence against oxidative protein damage. The present study further confirms the idea that the topical use of resveratrol can provide a good defence against CS-induced skin damage.

Cigarette smoking (CS) has been strongly linked to several health conditions including heart disease, lung cancer, and other respiratory and circulatory ailments. Deleterious effects of cigarette smoking on skin have also been well documented, but unlike effects on other organs, damage does not depend upon inhalation. The upper layer of the skin, the stratum corneum (SC) (rich in cholesterol fatty acids and ceramide) is very susceptible to damage induced by exposure to environmental stressors that can modify its lipid composition and thereby affect its function of protecting skin from dehydration. Scavenger Receptor B1 (SR-B1) is involved in uptake of cholesterol in several tissues including skin. We previously demonstrated that CS exposure induces formation of aldehydes (HNE) adducts that decrease SR-B1 expression. As topical resveratrol, a well-known polyphenolic stilbene, has been demonstrated to show benefits against skin disorders, we investigated its possible role as a protective agent against CS-induced reduction of SR-B1 expression in cutaneous tissue. In this study, we have demonstrated that resveratrol at the doses ranging from 0.5 to 10μM is not toxic, is able to increase SR-B1 protein levels in a dose-dependent manner in human keratinocytes. Moreover, when the cells that were pre-treated with different doses of resveratrol, were exposed to CS, the loss of SR-B1 was prevented in a dose dependent manner. In addition, in keratinocytes, resveratrol was also able to prevent an increase in HNE protein adducts induced by CS. In particular resveratrol was able to prevent HNE-SRB1 adducts formation. Thus, resveratrol appears to be a natural compound that could provide skin with a defense against exogenous stressors by protecting the essential cholesterol receptor, SR-B1.

Rett (RTT) syndrome is a severe neurological disorder that affects almost exclusively females. Several detectable mutations in the X-linked methyl-CpG-binding protein 2 gene (MECP2) are responsible for the onset of the disease. MeCP2 is a key transcription regulator involved in gene silencing via methylation-dependent remodeling of chromatin. Recent data highlight that lipid metabolism is perturbed in brains and livers of MECP2-null male mice. In addition, altered plasma lipid profile in RTT patients has been observed. Thus, the aim of the work is to investigate the protein network involved in cholesterol homeostasis maintenance on freshly isolated fibroblasts and plasma from both RTT and healthy donors. To this end, protein expression of 3-hydroxy-3methyl glutaryl Coenzyme A reductase (HMGR), sterol regulatory element binding proteins (SREBPs), low density lipoprotein receptor (LDLr) and scavenger receptor B-1 (SRB-1) was assessed in cultured skin fibroblasts from unaffected individuals and RTT patients. In addition, lipid profile and the abundance of proprotein convertase subtilisin/kexin type 9 (PCSK9) were analyzed on plasma samples. The obtained results demonstrate that the main proteins belonging to cholesterol regulatory network are altered in RTT female patients, providing the proof of principle that cholesterol metabolism may be taken into account as a new target for the treatment of specific features of RTT pathology.