Do meals contain a relevant amount of oxylipins? LC-MS-based analysis of oxidized fatty acids in food.

Several oxylipins are potent lipid mediators and are discussed to be absorbed after oral intake. However, information about their concentrations in oils and processed foods are scarce. Here, we analyzed the concentrations of mono-, di- and multihydroxy- as well as epoxy-PUFA in virgin and refined oils as well as in different foods/meals. Oil refining causes hydrolysis of epoxy-PUFA and thus high dihydroxy-PUFA concentrations (e.g. 15,16-DiHODE 290 µg/g in refined vs. 15 µg/g in virgin rapeseed oil), making the epoxy-to-diol ratio a potential marker for refined oils. Low oxylipin levels were found in foods with high amounts of saturated fatty acids such as Hamburger patties (around 30 µg/g). High concentrations (up to 1200 µg/g, 80 mg per serving) and high oxylipin/precursor-PUFA ratios were found in fried falafel and processed foods such as vegetarian sausage/fish fingers. Our study provides first insights in the oxylipin concentrations of our daily food, indicating a relevant intake.

Beyond Autoxidation and Lipoxygenases: Fatty Acid Oxidation Products in Plant Oils.

For decades, research on oxidation of linoleic acid (LA, C18:2 n6) and α-linolenic acid (ALA, C18:3 n3) in plant oils has focused on autoxidatively formed and lipoxygenase-derived 9-hydro(pero)xy- and 13-hydro(pero)xy-LA and -ALA. Here, using a non-targeted approach, we show that other hydroxy fatty acids are more abundant in plant oils. Liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry analyses unveiled highly abundant peaks in flaxseed and rapeseed oils. Using authentic reference standards, seven of the peaks were identified as 9-, 10-, 12-, 13-, and 15-HODE as well as 9- and 13-HOTrE. Additionally, six peaks were characterized based on the retention time, the exact mass of the [M-H]- ion, and its fragment ions as 16-OH-C18:3, 18-OH-C18:3, three isomers of 12-OH-C18:2, and one of 15-OH-C18:2. 16-OH-C18:3 and 18-OH-C18:3 were tentatively identified as 16-OH-ALA and 18-OH-ALA, respectively, based on autoxidation and terminal hydroxylation of ALA using CYP4F2. Investigation of formation pathways suggests that fatty acid desaturase 3 is involved in the formation of the 12-OH-C18:2 isomers, 15-HODE, and its isomer. The dominantly occurring 12-OH-C18:2 isomer was identified as 12R,S-OH-9Z,15Z-octadecadienoic acid (densipolic acid) based on a synthetic standard. The characterized oxylipins occurred in cold-pressed flaxseed and rapeseed oils at concentrations of up to 0.1 g/100 g and thus about sixfold higher than the well-known 9-hydro(pero)xy- and 13-hydro(pero)xy-LA and -ALA. Concentrations in sunflower oil were lower but increased when oil was pressed from preheated seeds. Overall, this study provides fundamental new information about the occurrence of oxidized fatty acids in plant oils, having the potential to characterize their quality and authenticity.

Trans-Hydroxy, Trans-Epoxy, and Erythro-dihydroxy Fatty Acids Increase during Deep-Frying.

Deep-frying of food is a common cooking technique causing thermal oxidation of fatty acids (FA). Here, we investigated for the first time the formation of hydroxy-, epoxy- and dihydroxy-FA derived from oleic, linoleic (LA), and α-linolenic acid (ALA) during frying. Potato chips were fried in high-oleic sunflower oil for 4 × 5 cycles on 2 days, and the oil was comprehensively analyzed by liquid chromatography-tandem mass spectrometry. During frying, the E,Z-9- and E,Z-13-hydroperoxy-LA and -ALA concentrations decrease while their corresponding hydroxy-FA remain constant. The concentrations of both E,E-9-/13-hydroperoxy-LA and E,E-9-/13-hydroxy-LA increase with the frying cycles, which is also found for the concentration of trans-epoxy-FA. The increase in trans-epoxy-FA is more pronounced than that of the corresponding cis-epoxy-FA, exceeding their concentrations on the second day of frying. This selective change in the cis-/trans-epoxy-FA ratio is also observed for their hydrolysis products: concentrations of erythro-dihydroxy-FA, derived from trans-epoxy-FA, increase during frying stronger than threo-dihydroxy-FA derived from cis-epoxy-FA. Based on these data, we suggest that the ratio of E,E-/E,Z-hydroxy-FA, in combination with the cis-/trans-epoxy-FA ratio, as well as the threo-/erythro-dihydroxy-FA ratio are promising new parameters to evaluate the heating of edible oils and to characterize the status of frying oils.

Weight-adjusted dosing of tinzaparin for thromboprophylaxis in obese medical patients.

Background: The optimal dose of tinzaparin for prophylaxis in obese medical patients is not well defined. Objectives: To evaluate the anti-Xa activity in obese medical patients on tinzaparin prophylaxis adjusted for actual bodyweight. Methods: Patients with a body mass index of ≥30 kg/m2 treated with 50 IU/kg tinzaparin once daily were prospectively included. Anti-Xa and anti-IIa activity; von Willebrand factor antigen and von Willebrand activity; factor VIII activity; D-dimer, prothrombin fragments; and thrombin generation were measured 4 hours after subcutaneous injection between days 1 and 14 after the initiation of tinzaparin prophylaxis. Results: We included 121 plasma samples from 66 patients (48.5% women), with a median weight of 125 kg (range, 82-300 kg) and a median body mass index of 41.9 kg/m2 (range, 30.1-88.6 kg/m2). The target anti-Xa activity of 0.2 to 0.4 IU/mL was achieved in 80 plasma samples (66.1%); 39 samples (32.2%) were below and 2 samples (1.7%) above the target range. The median anti-Xa activity was 0.25 IU/mL (IQR, 0.19-0.31 IU/mL), 0.23 IU/mL (IQR, 0.17-0.28 IU/mL), and 0.21 IU/mL (IQR, 0.17-0.25 IU/mL) on days 1 to 3, days 4 to 6, and days 7 to 14, respectively. The anti-Xa activity did not differ among the weight groups (P = .19). Injection into the upper arm compared to the abdomen resulted in a lower endogenous thrombin potential, a lower peak thrombin, and a trend to a higher anti-Xa activity. Conclusion: Dosing of tinzaparin adjusted for actual bodyweight in obese patients achieved anti-Xa activity in the target range for most patients, without accumulation or overdosing. In addition, there is a significant difference in thrombin generation depending on the injection site.

GC-MS analysis of oxysterols and their formation in cultivated liver cells (HepG2).

Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 µm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7ß-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.

Characterization of the Oxylipin Pattern and Other Fatty Acid Oxidation Products in Freshly Pressed and Stored Plant Oils.

Enzymatic and nonenzymatic oxidation of linoleic (LA) and α-linolenic acid (ALA) during pressing and storage of plant oils leads to a variety of oxylipins. We pressed oils from flaxseeds, rapeseeds, and sunflower seeds and analyzed the oxylipin pattern in freshly pressed oils. 9-/13-Hydro(pero)xy-LA/-ALA occurred in high concentration resulting probably from lipoxygenase-catalyzed reactions as well as autoxidation and photooxidation. However, in flaxseed and rapeseed oil, the highest concentrations were found for the terminal epoxy-ALA (15(16)-EpODE) and the hardly known 15-hydroxy-LA (15-HODE, 80 mg/100 g in flaxseed oil). Oils were stored for 6 months and the peroxide value (PV) as well as oxylipin and secondary volatile aldehyde concentrations were determined. While lipid peroxidation in flaxseed oil was surprisingly low, the oxylipin concentration and PV massively increased in rapeseed oil dependent on oxygen availability. Oxylipin concentrations correlated well with the PV, while secondary volatile aldehydes did not reflect the changes of oxylipins and PVs. The comprehensive analysis of hydroxy-, epoxy-, and dihydroxy-LA/-ALA reveals new and unique insights into the composition of plant oils and ongoing oxidation processes.

Comprehensive Analysis of Fatty Acid and Oxylipin Patterns in n3-PUFA Supplements.

Supplementing long-chain omega-3 polyunsaturated fatty acids (n3-PUFA) improves health. We characterized the pattern of total and non-esterified oxylipins and fatty acids in n3 supplements made of fish, krill, or micro-algae oil by LC-MS. All supplements contained the declared amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); however, their content per capsule and the concentration of other fatty acids varied strongly. Krill oil contained the highest total n3 oxylipin concentration (6000 nmol/g) and the highest degree of oxidation (EPA 0.7%; DHA 1.3%), while micro-algae oil (Schizochytrium sp.) showed the lowest oxidation (<0.09%). These oils contain specifically high amounts of the terminal hydroxylation product of EPA (20-HEPE, 300 nmol/g) and DHA (22-HDHA, 200 nmol/g), which can serve as an authenticity marker for micro-algae oil. Refined micro-algae and fish oil were characterized by NEFA levels of ≤0.1%. Overall, the oxylipin and fatty acid pattern allows gaining new insights into the origin and quality of n3-PUFA oils in supplements.

Fatty acid and oxylipin concentration differ markedly between different fetal bovine serums: A cautionary note.

Fetal bovine serum (FBS) has been used as a universal supplement in cell culture for more than six decades. This includes the investigation of lipid and lipid mediator formation and biology. Little is known about the (polyunsaturated) fatty acid composition and their oxidation products in FBS. Therefore, we analyzed six different FBS purchased from three different companies regarding their fatty acid and oxylipin concentrations. We found pronounced differences in the fatty acid concentrations. Even two batches of "standardized" FBS batches from one company showed drastic differences (e.g., for eicosapentaenoic acid 5 ± 1 µM vs. 11 ± 1 µM). Oxylipin concentrations also markedly differ between the FBS lots. The highest differences were found for 12-lipoxygenase products (e.g., 12-hydroxyeicosatetraenoic acid free 21-87 nM and total 58-108 nM), probably due to inconsistent serum generation procedures. Our results indicate that for cell culture studies dealing with lipid metabolism, researchers should carefully characterize their used FBS to ensure reliability and reproducibility of study outcomes.

Rapid quantification of fatty acids in plant oils and biological samples by LC-MS.

Analysis of fatty acids (FA) in food and biological samples such as blood is indispensable in modern life sciences. We developed a rapid, sensitive and comprehensive method for the quantification of 41 saturated and unsaturated fatty acids by means of LC-MS. Optimized chromatographic separation of isobaric analytes was carried out on a C8 reversed phase analytical column (100 × 2.1 mm, 2.6 µm core-shell particle) with a total run time of 15 min with back pressure lower than 300 bar. On an old triple quadrupole instrument (3200, AB Sciex), pseudo selected reaction monitoring mode was used for quantification of the poorly fragmenting FA, yielding limits of detection of 5-100 nM. Sample preparation was carried out by removal of phospholipids and triglycerides by solid-phase extraction (non-esterified fatty acids in oils) or saponification in iso-propanol (fatty acyls). This is not only a rapid strategy for quantification of fatty acyls, but allows the direct combination with the LC-MS-based analysis of fatty acid oxidation products (eicosanoids and other oxylipins) from the same sample. The concentrations of fatty acyls determined by means of LC-MS were consistent with those from GC-FID analysis demonstrating the accuracy of the developed method. Moreover, the method shows high precisions with a low intra-day (≤ 10% for almost all fatty acids in plasma and ≤ 15% in oils) and inter-day as well as inter-operator variability (< 20%). The method was successfully applied on human plasma and edible oils. The possibility to quantify non-esterified fatty acids in samples containing an excess of triacylglycerols and phospholipids is a major strength of the described approach allowing to gain new insights in the composition of biological samples.

A Walnut Diet in Combination with Enriched Environment Improves Cognitive Function and Affects Lipid Metabolites in Brain and Liver of Aged NMRI Mice.

This in vivo study aimed to test if a diet enriched with 6% walnuts alone or in combination with physical activity supports healthy ageing by changing the oxylipin profile in brain and liver, improving motor function, cognition, and cerebral mitochondrial function. Female NMRI mice were fed a 6% walnut diet starting at an age of 12 months for 24 weeks. One group was additionally maintained in an enriched environment, one group without intervention served as control. After three months, one additional control group of young mice (3 weeks old) was introduced. Motor and cognitive functions were measured using Open Field, Y-Maze, Rotarod and Passive Avoidance tests. Lipid metabolite profiles were determined using RP-LC-ESI(-)-MS/MS in brain and liver tissues of mice. Cerebral mitochondrial function was characterized by the determination of ATP levels, mitochondrial membrane potential and mitochondrial respiration. Expression of genes involved with mito- and neurogenesis, inflammation, and synaptic plasticity were determined using qRT-PCR. A 6% walnut-enriched diet alone improved spatial memory in a Y-Maze alternation test (p < 0.05) in mice. Additional physical enrichment enhanced the significance, although the overall benefit was virtually identical. Instead, physical enrichment improved motor performance in a Rotarod experiment (p* < 0.05) which was unaffected by walnuts alone. Bioactive oxylipins like hydroxy-polyunsaturated fatty acids (OH-PUFA) derived from linoleic acid (LA) were significantly increased in brain (p** < 0.01) and liver (p*** < 0.0001) compared to control mice, while OH-PUFA of α-linolenic acid (ALA) could only be detected in the brains of mice fed with walnuts. In the brain, walnuts combined with physical activity reduced arachidonic acid (ARA)-based oxylipin levels (p < 0.05). Effects of walnut lipids were not linked to mitochondrial function, as ATP production, mitochondrial membrane potential and mitochondrial respiration were unaffected. Furthermore, common markers for synaptic plasticity and neuronal growth, key genes in the regulation of cytoprotective response to oxidative stress and neuronal growth were unaffected. Taken together, walnuts change the oxylipin profile in liver and brain, which could have beneficial effects for healthy ageing, an effect that can be further enhanced with an active lifestyle. Further studies may focus on specific nutrient lipids that potentially provide preventive effects in the brain.

Stability of oxylipins during plasma generation and long-term storage.

Oxidized unsaturated fatty acids - i.e. eicosanoids and other oxylipins - are lipid mediators involved in the regulation of numerous physiological functions such as inflammation, blood coagulation, vascular tone and endothelial permeability. They have raised strong interest in clinical lipidomics in order to understand their role in health and diseases and their use as biomarkers. However, before the clinical translation, it is crucial to validate the analytical reliability of oxylipins. This notably requires to assess the putative artificial formation or degradation of oxylipins by (unsuitable) blood handling during plasma generation, storage and sample preparation. Using a liquid chromatography-mass spectrometry method covering 133 oxylipins we comprehensively analyzed the total (free + esterified) oxylipin profile in plasma and investigated the influence of i) addition of additives during sample preparation, ii) different storage times and temperatures during the transitory stage of plasma generation and iii) long-term storage of plasma samples at -80 °C. Addition of radical scavenger butylated hydroxytoluene reduced the apparent concentrations of hydroxy-PUFA and thus should be added to the samples at the beginning of sample preparation. The concentrations of all oxylipin classes remained stable (within analytical variance of 20%) during the transitory stage of plasma generation up to 24 h at 4 °C or 4 h at 20 °C before centrifugation of EDTA-whole blood and up to 5 days at -20 °C after plasma separation. The variations in oxylipin concentrations did not correlate with storage time, storage temperature or stage of plasma generation. A significant increase of potentially lipoxygenase derived hydroxy-PUFA compared to immediate processing was only detected when samples were stored for longer times before centrifugation, plasma separation as well as freezing of plasma revealing residual enzymatic activity. Autoxidative rather than enzymatic processes led to a slightly increased concentration of 9-HETE when plasma samples were stored at -80 °C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 °C supporting the application of oxylipin profiling in clinical research.

Targeting esterified oxylipins by LC-MS - Effect of sample preparation on oxylipin pattern.

A major part of oxygenated metabolites of polyunsaturated fatty acids - i.e. eicosanoids and other oxylipins - in biological samples is found in the esterified form. Yet, their biological role is only poorly understood. For quantification of esterified oxylipins in biological samples current protocols mostly apply alkaline hydrolysis with or without prior lipid extraction to release oxylipins into their free form which can be subsequently quantified via liquid chromatography-mass spectrometry. Herein, a detailed protocol for precise and reproducible quantification of esterified oxylipins in plasma is presented comprising i) extraction of lipids and removal of proteins with iso-propanol, ii) base hydrolysis with potassium hydroxide to saponify lipids and iii) solid phase extraction of the liberated oxylipins on C8/anion exchange mixed mode material. Unequal extraction of internal standards and lipid classes during lipid extraction before hydrolysis led to distorted concentrations, emphasizing that the choice of solvent used in this step is important to minimize discrimination. Regarding the hydrolysis conditions, at least 30 min incubation at 60 °C is required with 0.1 M KOH in sample. Drying of the SPE cartridges is a critical parameter since autoxidation processes of PUFA, which are present in high concentrations after cleavage, lead to artificial formation of epoxy fatty acids. With the developed protocol, inter-day, intra-day and inter-operator variance was <21% for most oxylipins including hydroxy-, dihydroxy-, and epoxy-PUFA. The applicability of the developed methodology is demonstrated by investigating the changes in the oxylipin pattern following omega-3 fatty acid feeding to rats.

Single-Dose SDA-Rich Echium Oil Increases Plasma EPA, DPAn3, and DHA Concentrations.

The omega-3 (n3) polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with health benefits. The primary dietary source of EPA and DHA is seafood. Alpha-linoleic acid (ALA) has not been shown to be a good source for EPA and DHA; however, stearidonic acid (SDA)-which is naturally contained in echium oil (EO)-may be a more promising alternative. This study was aimed at investigating the short-term n3 PUFA metabolism after the ingestion of a single dose of EO. Healthy young male subjects (n = 12) ingested a single dose of 26 g of EO after overnight fasting. Plasma fatty acid concentrations and relative amounts were determined at baseline and 2, 4, 6, 8, 24, 48, and 72 h after the ingestion of EO. During the whole examination period, the participants received standardized nutrition. Plasma ALA and SDA concentrations increased rapidly after the single dose of EO. Additionally, EPA and DPAn3 concentrations both increased significantly by 47% after 72 h compared to baseline; DHA concentrations also significantly increased by 21% after 72 h. To conclude, EO increases plasma ALA, SDA, EPA, DPAn3, and DHA concentrations and may be an alternative source for these n3 PUFAs.

Pan European Phenological database (PEP725): a single point of access for European data.

The Pan European Phenology (PEP) project is a European infrastructure to promote and facilitate phenological research, education, and environmental monitoring. The main objective is to maintain and develop a Pan European Phenological database (PEP725) with an open, unrestricted data access for science and education. PEP725 is the successor of the database developed through the COST action 725 "Establishing a European phenological data platform for climatological applications" working as a single access point for European-wide plant phenological data. So far, 32 European meteorological services and project partners from across Europe have joined and supplied data collected by volunteers from 1868 to the present for the PEP725 database. Most of the partners actively provide data on a regular basis. The database presently holds almost 12 million records, about 46 growing stages and 265 plant species (including cultivars), and can be accessed via http://www.pep725.eu/ . Users of the PEP725 database have studied a diversity of topics ranging from climate change impact, plant physiological question, phenological modeling, and remote sensing of vegetation to ecosystem productivity.

Nonmelanoma skin cancer in mountain guides: high prevalence and lack of awareness warrant development of evidence-based prevention tools.

AIMS OF THE STUDY: Nonmelanoma skin cancer (NMSC) is the most common cancer in Switzerland and Europe. The main causative factor is exposure to ultraviolet radiation, which puts outdoor workers in general at a higher risk of developing NMSC than indoor workers. However, few studies have clinically examined the risk of developing NMSC to outdoor workers, especially mountain guides. We aimed to investigate the prevalence of NMSC and corresponding precancerous lesions, and the associated risk behaviour of mountain and ski guides in order to develop future prevention programmes. METHODS: We conducted a cross-sectional study including mountain and ski guides from southern Germany, who underwent a full-body skin check-up by a dermatologist. We assessed their NMSC awareness and risk behaviour using a paper-based questionnaire. RESULTS: Of the 62 state-certified mountain and ski guides (55 men, 7 women; mean age 52.9 ± 13.4 years) included in this study, 27 (43.5%) were diagnosed with NMSC or its premalignant stages. In addition, 59.7% of the participants expressed the opinion that their protection from ultraviolet radiation exposure needs to be improved; 83.6% requested further information on NMSC, and 48.5% had never undergone a skin check-up or consulted a dermatologist before. CONCLUSIONS: Mountain and ski guides are at a high risk for developing NMSC. Their unmet medical needs indicate an underestimation of NMSC prevalence, which is usually based on reports by insurance companies, and offer the chance for developing evidence-based awareness and prevention tools that can be promoted to individuals with other outdoor jobs.

Use of the GlideScope Ranger Video Laryngoscope for Emergency Intubation in the Prehospital Setting: A Randomized Control Trial.

OBJECTIVES: We sought to assess whether the GlideScope Ranger video laryngoscope may be a reliable alternative to direct laryngoscopy in the prehospital setting. DESIGN: Multicenter, prospective, randomized, control trial with patient recruitment over 18 months. SETTING: Four study centers operating physician-staffed rescue helicopters or ground units in Austria and Norway. PATIENTS: Adult emergency patients requiring endotracheal intubation. INTERVENTIONS: Airway management strictly following a prehospital algorithm. First and second intubation attempt employing GlideScope or direct laryngoscopy as randomized; third attempt crossover. After three failed intubation attempts, immediate use of an extraglottic airway device. MEASUREMENTS AND MAIN RESULTS: A total of 326 patients were enrolled. Success rate with the GlideScope (n = 168) versus direct laryngoscopy (n = 158) group was 61.9% (104/168) versus 96.2% (152/158), respectively (p < 0.001). The main reasons for failed GlideScope intubation were failure to advance the tube into the larynx or trachea (26/168 vs 0/158; p < 0.001) and/or impaired sight due to blood or fluids (21/168 vs 3/158; p < 0.001). When GlideScope intubation failed, direct laryngoscopy was successful in 61 of 64 patients (95.3%), whereas GlideScope enabled intubation in four of six cases (66.7%) where direct laryngoscopy failed (p = 0.055). In addition, GlideScope was prone to impaired visualization of the monitor because of ambient light (29/168; 17.3%). There was no correlation between success rates and body mass index, age, indication for airway management, or experience of the physicians, respectively. CONCLUSIONS: Video laryngoscopy is an established tool in difficult airway management, but our results shed light on the specific problems in the emergency medical service setting. Prehospital use of the GlideScope was associated with some major problems, thus resulting in a lower intubation success rate when compared with direct laryngoscopy.

The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular haemostasis in TNFα -induced inflammation in vivo.

INTRODUCTION: Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα -induced endothelial inflammation in vivo. METHODS: Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. RESULTS: Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα -induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα -induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα -induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. CONCLUSIONS: The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF α -induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.

AIMS: The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. METHODS AND RESULTS: ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. CONCLUSION: Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.