A combined targeted/untargeted LC-MS/MS-based screening approach for mammalian cell lines treated with ionic liquids : Toxicity correlates with metabolic profile

This work presents the development and validation of a quantitative HILIC UHPLC-ESI-QTOF-MS/MS method for amino acids combined with untargeted metabolic profiling of human corneal epithelial (HCE) cells after treatment with ionic liquids. The work included a preliminary metabotoxicity screening of 14 different ionic liquids, of which 9 carefully selected ionic liquids were chosen for a metabolomics study. This study is focused on the correlation between the toxicity of the ionic liquids and their metabolic profiles. The method development included the comparison of different MS/MS acquisition modes. A sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) method with variable Q1 window widths and narrow Q1 target windows of 5 Da for most of the amino acids was selected as the optimal acquisition mode. Due to the absence of a true blank matrix, C-13,N-15-isotopically labelled amino acids were utilized as surrogate calibrants, instead of proteinogenic amino acids. Partial least squares (PLS) analysis of the median effective concentrations (EC50) of 9 selected ionic liquids showed a correlation with their metabolic profile measured by the untargeted screening.Peer reviewe

Sex Steroid Hormone Analysis in Human Tear Fluid Using a Liquid Chromatography—Mass Spectrometry Method

The marked sexual dimorphism prevalent in inflammatory/autoimmune diseases is mostly due to sex hormone actions. One common eye disease that disproportionately affects women is dry eye. Thus, our aim was to optimise our highly sensitive liquid chromatography–tandem mass spectrometry method for steroid hormone quantification in tear fluid (TF). We used tears and matched serum samples from 10 heathy individuals. Estrone, estradiol testosterone, progesterone, androstenedione, and dehydroepiandrosterone, were quantified with an HPLC coupled with a Triple Quad 5500 MS. Estrone was measured in 80% of female and 20% of male TF samples (mean ± SD, 68.9 ± 62.2 pmol/L), whereas estradiol was undetectable in tears. Progesterone was identified in half of the female tear samples (2.91 ± 3.47 nmol/L) but in none of the male samples, whereas testosterone was quantifiable only in male tears (0.24 ± 0.1 nmol/L). TF hormone levels were, on average, from 1.4% to 55% of systemic values. Estrone, progesterone, and testosterone levels in tears correlated with the matching serum samples (r = 0.82, 0.79, and 0.85, respectively), but androstenedione and dehydroepiandrosterone showed no correlations. Our LC–MS/MS method could detect five out of the six steroid hormones studied in individual human TF samples and could therefore be used to analyse the role of sex steroids in eye diseases

Hyperosmolarity induced lipid droplet formation depends on ceramide production by neutral sphingomyelinase 2

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Interaction of phospholipid transfer protein with human tear fluid mucins

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Correlation between Ionic Liquid Cytotoxicity and Liposome-Ionic Liquid Interactions

This study aims at extending the understanding of the toxicity mechanism of ionic liquids (ILs) using various analytical methods and cytotoxicity assays. The cytotoxicity of eight ILs and one zwitterionic compound was determined using mammalian and bacterial cells. The time dependency of the IL toxicity was assessed using human corneal epithelial cells. Hemolysis was performed using human red blood cells and the results were compared with destabilization data of synthetic liposomes upon addition of ILs. The effect of the ILs on the size and zeta potential of liposomes revealed information on changes in the lipid bilayer. Differential scanning calorimetry was used to study the penetration of the ILs into the lipid bilayer. Pulsed field gradient nuclear magnetic resonance spectroscopy was used to determine whether the ILs occurred as unimers, micelles, or if they were bound to liposomes. The results show that the investigated ILs can be divided into three groups based on the cytotoxicity mechanism: cell wall disrupting ILs, ILs exerting toxicity through both cell wall penetration and metabolic alteration, and ILs affecting solely on cell metabolism.Peer reviewe

Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet

Objective: Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown. Methods: K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3eIg in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments. Results: We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity. Conclusions: These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance. (C) 2014 The Authors. Published by Elsevier GmbH.Peer reviewe

Environmental stress and the corneal epithelium

The cornea is the first optical element of the eye and, together with the eyelids, eye socket, tears, and sclera, shares an important part in ocular protection. It is a thin, transparent, avascular tissue with a rigorous layered structure. The continuous contact with the outside environment exposes the ocular surface tissues, such as the corneal epithelium, to pathogens, mechanical traumas, irritants, toxins, allergens, or radiation from the sun. The cellular stress response represents an adaptive reaction to environmental stimuli and defines the health-state of the tissue, the absence/presence of clinical manifestations. We have aimed in this thesis project to study the stress response of the corneal epithelium to environmental stimuli and to determine its contribution to ocular surface diseases such as climatic droplet keratopathy (CDK), infection, or dry eye disease. A cell culture model of the corneal epithelium was exposed to environmental stress – UV radiation, LPS, or hyperosmolarity (HO) – to identify macromolecular alterations: mRNA expression, protein localization, enzyme activation, lipid conversions. CDK is a degenerative disease of the cornea with increased prevalence in warm, dry climate. Examination of corneal tissue and tears from patients with CDK suggested an involvement of metalloproteinases (MMPs) in the disease-associated tissue degradation. Our cellular model helped reveal the connexion between UV radiation and the unbalanced secretion of gelatinases (MMP-2 and MMP-9) and thus explain in part the pathogenesis of this rare disease. The evaluation of the inflammatory response to UV, initially, and then to LPS, or HO, highlighted IL-8 secretion as an acute stress marker and followed throughout the studies. Human corneal epithelial cells were found also to release lipid-modifying enzymes into the cell culture medium as a response to stress. Of particular importance to us were the enzymes of the sphingolipid metabolism, a lipid signalling pathway of great importance in the stress response. These enzymes were released as part of cell-derived extracellular vesicles, the vesicle-lipids, however, were the mediators of a significant decrease in IL-8 levels. The same sphingolipid enzymes appeared responsible for the intracellular response to HO, controlling the IL-8 production but also the stress-induced neutral lipid loading. We have therefore succeeded to establish a causative link between UV radiation and tissue degeneration in CDK, to determine the role of the sphingolipid signalling pathway in ocular surface stress and to discover more about HO consequences in the corneal epithelium. The stress response at the ocular surface is a thin balance between tissue protection and maintenance of function. Inflammation represents one of the most relevant clinical signs of distress and we aimed to identify targets for therapies that seek to restore tissue homeostasis.Silmän sarveiskalvo toimii tärkeässä roolissa suojauduttaessa ulkoisilta silmään kohdistuvilta stressitekijöiltä. Sarveiskalvo altistuu jatkuvasti erilaisille patogeeneille, mekaaniselle stressille, kemiallisille ärsykkeille, allergeeneille ja auringon UV-säteilylle. Altistuksen yhteydessä solut adaptiivisesti reagoivat stressiin pyrkien korjaamaan sen spesifisillä mekanismeilla. Tämän väitöstyön tavoitteena on ollut selvittää sarveiskalvon epiteelisoluviljelmää käyttäen kolmen erilaisen ulkoisen stressitekijän (UV-säteily, endotoksiini eli LPS-käsittely ja korkea suolapitoisuus eli hyperosmolariteetti) vaikutusta solujen tasapainoon ja onko saaduilla tuloksilla yhteyttä kliinisesti tärkeisiin silmän häiriötiloihin, joista tärkeimpinä tässä yhteydessä ovat CDK-keratopatia, infektiot ja kuivasilmäisyys. CDK-potilailta tutkitut sarveiskalvo- ja kyynelnäytteet osoittivat, että metalloproteinaasit (MMP) ovat tärkeitä tautiin liittyvässä sarveiskalvon soludegradaatiossa. Käytössä ollut sarveiskalvon epiteelisoluviljelymalli osoitti, että UV-säteilyn ja erityisesti MMP-2 ja MMP-9 entsyymien soluerityksen välillä oli selkeä yhteys ja liittyy osaltaan CDK:n patogeneesiin. UV-, LPS- ja hyperosmolariteetti-käsittelyjen aikana sarveiskalvon epiteelisolut erittivät interleukiini 8:aa (IL-8), ja tästä sytokiinistä muodostuikin tärkein akuutin stressin biomarkkeri soluviljelykokeissa. Erityisen tärkeä havainto lisäksi oli, että epiteelisolut vasteena stressiin erittivät ekstrasellulaari-vesikkeleitä, jotka sisälsivät sfingolipidi-aineenvaihduntaan osallistuvia entsyymejä. On muistettava, että solujen sfingolipidien signalointireitit ovat erittäin tärkeitä stressivasteen yhteydessä. Yhteenvetona voidaan todeta, että väitöstyössä on osoitettu kausaalinen yhteys UV-säteilyn ja CDK:hon liittyvän sarveiskalvovaurion välillä ja osoitettu sfingolipidien signaloinnin liittyminen sarveiskalvoepiteelin stressiin. Kokonaisuudessaan työssä on pyritty identifioimaan terapian kannalta tärkeitä molekyylejä ja metaboliareittejä, joihin vaikuttamalla voidaan silmän sarveiskalvon tulehduksellista stressitilaa lieventää ja näin ollen tulokset ovat kliinisesti merkittäviä