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Controlled delivery of corticosteroids using nanoparticles to the skin and corneal epithelium may reduce their side effects and maximize treatment effectiveness. Dexamethasone-loaded ethyl cellulose, Eudragit® RS and ethyl cellulose/Eudragit® RS nanoparticles were prepared by the solvent evaporation method. Dexamethasone release from the polymeric nanoparticles was investigated in vitro using Franz diffusion cells. Drug penetration was also assessed ex vivo using excised human skin. Nanoparticle toxicity was determined by MTT and H2DCFDA assays. Eudragit® RS nanoparticles were smaller and positively charged but had a lower dexamethasone loading capacity (0.3–0.7%) than ethyl cellulose nanoparticles (1.4–2.2%). By blending the two polymers (1:1), small (105 nm), positively charged (+37 mV) nanoparticles with sufficient dexamethasone loading (1.3%) were obtained. Dexamethasone release and penetration significantly decreased with decreasing drug to polymer ratio and increased when Eudragit® RS was blended with ethyl cellulose. Ex vivo, drug release and penetration from the nanoparticles was slower than a conventional cream. The nanoparticles bear no toxicity potentials except ethyl cellulose nanoparticles had ROS generation potential at high concentration. In conclusion, the nanoparticles showed great potential to control the release and penetration of corticosteroids on the skin and mucus membrane and maximize treatment effectiveness.
Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets
(2017)
Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.
Background: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study.
Methods and Results: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT.
Conclusions: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage.
Enhanced topical delivery of dexamethasone by beta-cyclodextrin decorated thermoresponsive nanogels
(2018)
Highly hydrophilic, responsive nanogels are attractive as potential systems for the topical delivery of bioactives encapsulated in their three-dimensional polymeric scaffold. Yet, these drug carrier systems suffer from drawbacks for efficient delivery of hydrophobic drugs. Addressing this, β-cyclodextrin (βCD) could be successfully introduced into the drug carrier systems by exploiting its unique affinity toward dexamethasone (DXM) as well as its role as topical penetration enhancer. The properties of βCD could be combined with those of thermoresponsive nanogels (tNGs) based on dendritic polyglycerol (dPG) as a crosslinker and linear thermoresponsive polyglycerol (tPG) inducing responsiveness to temperature changes. Electron paramagnetic resonance (EPR) studies localized the drug within the hydrophobic cavity of βCD by differences in its mobility and environmental polarity. In fact, the fabricated carriers combining a particulate delivery system with a conventional penetration enhancer, resulted in an efficient delivery of DXM to the epidermis and the dermis of human skin ex vivo (enhancement compared to commercial DXM cream: ∼2.5 fold in epidermis, ∼30 fold in dermis). Furthermore, DXM encapsulated in βCD tNGs applied to skin equivalents downregulated the expression of proinflammatory thymic stromal lymphopoietin (TSLP) and outperformed a commercially available DXM cream.
Two decades ago, sphingosine 1-phosphate (S1P) was discovered as a novel bioactive molecule that regulates a variety of cellular functions. The plethora of S1P-mediated effects is due to the fact that the sphingolipid not only modulates intracellular functions but also acts as a ligand of G protein-coupled receptors after secretion into the extracellular environment. In the plasma, S1P is found in high concentrations, modulating immune cell trafficking and vascular endothelial integrity. The liver is engaged in modulating the plasma S1P content, as it produces apolipoprotein M, which is a chaperone for the S1P transport. Moreover, the liver plays a substantial role in glucose and lipid homeostasis. A dysfunction of glucose and lipid metabolism is connected with the development of liver diseases such as hepatic insulin resistance, non-alcoholic fatty liver disease, or liver fibrosis. Recent studies indicate that S1P is involved in liver pathophysiology and contributes to the development of liver diseases. In this review, the current state of knowledge about S1P and its signaling in the liver is summarized with a specific focus on the dysregulation of S1P signaling in obesity-mediated liver diseases. Thus, the modulation of S1P signaling can be considered as a potential therapeutic target for the treatment of hepatic diseases.
Owing their unique chemical and physical properties core-multishell (CMS) nanocarriers are thought to underlie their exploitable biomedical use for a topical treatment of skin diseases. This highlights the need to consider not only the efficacy of CMS nanocarriers but also the potentially unpredictable and adverse consequences of their exposure thereto. As CMS nanocarriers are able to penetrate into viable layers of normal and stripped human skin ex vivo as well as in in vitro skin disease models the understanding of nanoparticle crosstalk with components of the immune system requires thorough investigation. Our studies highlight the biocompatible properties of CMS nanocarriers on Langerhans cells of the skin as they did neither induce cytotoxicity and genotoxicity nor cause reactive oxygen species (ROS) or an immunological response. Nevertheless, CMS nanocarriers were efficiently taken up by Langerhans cells via divergent endocytic pathways. Bioimaging of CMS nanocarriers by fluorescence lifetime imaging microscopy (FLIM) and flow cytometry indicated not only a localization within the lysosomes but also an energy-dependent exocytosis of unmodified CMS nanocarriers into the extracellular environment. (C) 2018 Elsevier Ltd. All rights reserved.
Aims/hypothesis Low-protein diets are well known to improve glucose tolerance and increase energy expenditure. Increases in circulating fibroblast growth factor 21 (FGF21) have been implicated as a potential underlying mechanism. Methods We aimed to test whether low-protein diets in the context of a high-carbohydrate or high-fat regimen would also protect against type 2 diabetes in New Zealand Obese (NZO) mice used as a model of polygenetic obesity and type 2 diabetes. Mice were placed on high-fat diets that provided protein at control (16 kJ%; CON) or low (4 kJ%; low-protein/high-carbohydrate [LP/HC] or low-protein/high-fat [LP/HF]) levels. Results Protein restriction prevented the onset of hyperglycaemia and beta cell loss despite increased food intake and fat mass. The effect was seen only under conditions of a lower carbohydrate/fat ratio (LP/HF). When the carbohydrate/fat ratio was high (LP/HC), mice developed type 2 diabetes despite the robustly elevated hepatic FGF21 secretion and increased energy expenditure. Conclusion/interpretation Prevention of type 2 diabetes through protein restriction, without lowering food intake and body fat mass, is compromised by high dietary carbohydrates. Increased FGF21 levels and elevated energy expenditure do not protect against hyperglycaemia and type 2 diabetes per se.
Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.
Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.
Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.
Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.
Studies over the past several years have demonstrated the important role of sphingolipids in cystic fibrosis (CF), chronic obstructive pulmonary disease and acute lung injury. Ceramide is increased in airway epithelial cells and alveolar macrophages of CF mice and humans, while sphingosine is dramatically decreased. This increase in ceramide results in chronic inflammation, increased death of epithelial cells, release of DNA into the bronchial lumen and thereby an impairment of mucociliary clearance; while the lack of sphingosine in airway epithelial cells causes high infection susceptibility in CF mice and possibly patients. The increase in ceramide mediates an ectopic expression of beta 1-integrins in the luminal membrane of CF epithelial cells, which results, via an unknown mechanism, in a down-regulation of acid ceramidase. It is predominantly this down-regulation of acid ceramidase that results in the imbalance of ceramide and sphingosine in CF cells. Correction of ceramide and sphingosine levels can be achieved by inhalation of functional acid sphingomyelinase inhibitors, recombinant acid ceramidase or by normalization of beta 1-integrin expression and subsequent re-expression of endogenous acid ceramidase. These treatments correct pulmonary inflammation and prevent or treat, respectively, acute and chronic pulmonary infections in CF mice with Staphylococcus aureus and mucoid or non-mucoid Pseudomonas aeruginosa. Inhalation of sphingosine corrects sphingosine levels only and seems to mainly act against the infection. Many antidepressants are functional inhibitors of the acid sphingomyelinase and were designed for systemic treatment of major depression. These drugs could be repurposed to treat CF by inhalation.
Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.
Major depressive disorder (MDD) is a common and severe disease characterized by mood changes, somatic alterations, and often suicide. MDD is treated with antidepressants, but the molecular mechanism of their action is unknown. We found that widely used antidepressants such as amitriptyline and fluoxetine induce autophagy in hippocampal neurons via the slow accumulation of sphingomyelin in lysosomes and Golgi membranes and of ceramide in the endoplasmic reticulum (ER). ER ceramide stimulates phosphatase 2A and thereby the autophagy proteins Ulk, Beclin, Vps34/Phosphatidylinositol 3-kinase, p62, and Lc3B. Although treatment with amitriptyline or fluoxetine requires at least 12 days to achieve sphingomyelin accumulation and the subsequent biochemical and cellular changes, direct inhibition of sphingomyelin synthases with tricyclodecan-9-yl-xanthogenate (D609) results in rapid (within 3 days) accumulation of ceramide in the ER, activation of autophagy, and reversal of biochemical and behavioral signs of stress-induced MDD. Inhibition of Beclin blocks the antidepressive effects of amitriptyline and D609 and induces cellular and behavioral changes typical of MDD. These findings identify sphingolipid-controlled autophagy as an important target for antidepressive treatment methods and provide a rationale for the development of novel antidepressants that act within a few days.
Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) lead to metabolic alterations and a sustained formation of 2-hydroxyglutarate (2-HG). 2-HG is an oncometabolite as it inhibits the activity of alpha-ketoglutarate-dependent dioxygenases such as ten-eleven translocation (TET) enzymes. Inhibitors of mutant IDH enzymes, like ML309, are currently tested in order to lower the levels of 2-HG. Vitamin C (VC) is an inducer of TET enzymes. To test a new therapeutic avenue of synergistic effects, the anti-neoplastic activity of inhibition of mutant IDH1 via ML309 in the presence of VC was investigated in the colon cancer cell line HCT116 IDH1(R132H/+) (harbouring a mutated IDH1 allele) and the parental cells HCT116 IDH1(+/+) (wild type IDH1). Measurement of the oncometabolite indicated a 56-fold higher content of 2-HG in mutated cells compared to wild type cells. A significant reduction of 2-HG was observed in mutated cells after treatment with ML 309, whereas VC produced only minimally changes of the oncometabolite. However, combinatorial treatment with both, ML309 and VC, in mutated cells induced pronounced reduction of 2-HG leading to levels comparable to those in wild type cells. The decreased level of 2-HG in mutated cells after combinatorial treatment was accompanied by an enhanced global DNA hydroxymethylation and an increased gene expression of certain tumour suppressors. Moreover, mutated cells showed an increased percentage of apoptotic cells after treatment with non-cytotoxic concentrations of ML309 and VC. These results suggest that combinatorial therapy is of interest for further investigation to rescue TET activity and treatment of IDH1/2 mutated cancers.
Improved knowledge of retinoblastoma chemotherapy resistance is needed to raise treatment efficiency. The objective of this study was to test whether etoposide alters glucosyl-ceramide, ceramide, sphingosine, and sphingosine-1-phosphate (sphingosine-1-P) levels in parental retinoblastoma cells (WERI Rb1) or their etoposide-resistant subclones (WERI EtoR). WERI Rb1 and WERI EtoR were incubated with 400 ng/ml etoposide for 24 h. Levels of glucosyl-ceramides, ceramides, sphingosine, sphingosine-1-P were detected by Q-TOF mass spectrometry. Statistical analysis was done by ANOVA followed by Tukey post-hoc test (p < 0.05). The mRNA expression of sphingolipid pathways enzymes in WERI Rb1, WERI EtoR and four human retinoblastoma tissue samples was analyzed by quantitative real-time PCR. Pathways enzymes mRNA expression confirmed similarities of human sphingolipid metabolism in both cell lines and tissue samples, but different relative expression. Significant up-regulation of sphingosine was seen in both cell lines (p < 0.001). Only sphingosine-1-P up-regulation was significantly increased in WERI EtoR (p < 0.01), but not in WERI Rb1 (p > 0.2). Both cell lines upregulate pro-apoptotic sphingosine after etoposide incubation, but only WERI EtoR produces additional survival favorable sphingosine-1-P. These data may suggest a role of sphingosine-1-P in retinoblastoma chemotherapy resistance, although this seems not to be the only resistance mechanism.
This case report addresses the problem of underreporting negative results and adverse side effects in animal testing. We present our findings regarding a hyperphagic mouse model associated with unforeseen high mortality. The results outline the necessity of reporting detailed information in the literature to avoid duplication. Obese mouse models are essential in the study of obesity, metabolic syndrome and diabetes mellitus. An experimental model of obesity can be induced by the administration of gold thioglucose (GTG). After transcending the blood-brain barrier, the GTG molecule interacts with regions of the ventromedial hypothalamus, thereby primarily targeting glucose-sensitive neurons. When these neurons are impaired, mice become insensitive to the satiety effects of glucose and develop hyperphagia. In a pilot study for optimising dosage and body weight development, C57BL/6 mice were treated with GTG (0.5 mg/g body weight) or saline, respectively. Animals were provided a physiological amount of standard diet (5 g per animal) for the first 24 hours after treatment to prevent gastric dilatation. Within 24 hours after GTG injection, all GTG-treated animals died of gastric overload and subsequent circulatory shock. Animals developed severe attacks of hyperphagia, and as the amount of provided chow was restricted, mice exhibited unforeseen pica and ingested bedding material. These observations strongly suggest that restricted feeding is contraindicated concerning GTG application. Presumably, the impulse of excessive food intake was a strong driving force. Therefore, the actual degree of suffering in the GTG-induced model of hyperphagia should be revised from moderate to severe.