MacroH2A1.1 as a crossroad between epigenetics, inflammation and metabolism of mesenchymal stromal cells in myelodysplastic syndromes.

Ineffective hematopoiesis is a hallmark of myelodysplastic syndromes (MDS). Hematopoietic alterations in MDS patients strictly correlate with microenvironment dysfunctions, eventually affecting also the mesenchymal stromal cell (MSC) compartment. Stromal cells are indeed epigenetically reprogrammed to cooperate with leukemic cells and propagate the disease as "tumor unit"; therefore, changes in MSC epigenetic profile might contribute to the hematopoietic perturbations typical of MDS. Here, we unveil that the histone variant macroH2A1 (mH2A1) regulates the crosstalk between epigenetics and inflammation in MDS-MSCs, potentially affecting their hematopoietic support ability. We show that the mH2A1 splicing isoform mH2A1.1 accumulates in MDS-MSCs, correlating with the expression of the Toll-like receptor 4 (TLR4), an important pro-tumor activator of MSC phenotype associated to a pro-inflammatory behavior. MH2A1.1-TLR4 axis was further investigated in HS-5 stromal cells after ectopic mH2A1.1 overexpression (mH2A1.1-OE). Proteomic data confirmed the activation of a pro-inflammatory signature associated to TLR4 and nuclear factor kappa B (NFkB) activation. Moreover, mH2A1.1-OE proteomic profile identified several upregulated proteins associated to DNA and histones hypermethylation, including S-adenosylhomocysteine hydrolase, a strong inhibitor of DNA methyltransferase and of the methyl donor S-adenosyl-methionine (SAM). HPLC analysis confirmed higher SAM/SAH ratio along with a metabolic reprogramming. Interestingly, an increased LDHA nuclear localization was detected both in mH2A1.1-OE cells and MDS-MSCs, probably depending on MSC inflammatory phenotype. Finally, coculturing healthy mH2A1.1-OE MSCs with CD34+ cells, we found a significant reduction in the number of CD34+ cells, which was reflected in a decreased number of colony forming units (CFU-Cs). These results suggest a key role of mH2A1.1 in driving the crosstalk between epigenetic signaling, inflammation, and cell metabolism networks in MDS-MSCs.

Cerebrospinal fluid ATP metabolites in multiple sclerosis.

Increased axonal energy demand and mitochondrial failure have been suggested as possible causes for axonal degeneration and disability in multiple sclerosis. Our objective was to test whether ATP depletion precedes clinical, imaging and biomarker evidence for axonal degeneration in multiple sclerosis. The method consisted of a longitudinal study which included 21 patients with multiple sclerosis. High performance liquid chromatography was used to quantify biomarkers of the ATP metabolism (oxypurines and purines) from the cerebrospinal fluid at baseline. The Expanded Disability Status Scale, MRI brain imaging measures for brain atrophy (ventricular and parenchymal fractions), and cerebrospinal fluid biomarkers for axonal damage (phosphorylated and hyperphosphorylated neurofilaments) were quantified at baseline and 3-year follow-up. Central ATP depletion (sum of ATP metabolites >19.7 micromol/litre) was followed by more severe progression of disability if compared to normal ATP metabolites (median 1.5 versus 0, p< 0.05). Baseline ATP metabolite levels correlated with change of Expanded Disability Status Scale in the pooled cohort (r= 0.66, p= 0.001) and subgroups (relapsing-remitting patients: r= 0.79, p< 0.05 and secondary progressive/primary progressive patients: r= 0.69, p< 0.01). There was no relationship between central ATP metabolites and either biomarker or MRI evidence for axonal degeneration. The data suggests that an increased energy demand in multiple sclerosis may cause a quantifiable degree of central ATP depletion. We speculate that the observed clinical disability may be related to depolarisation associated conduction block.

Modulation of AQP4 expression by the protein kinase C activator, phorbol myristate acetate, decreases ischemia-induced brain edema.

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), is known to interact with aquaporin-4 (AQP4), a water-selective transporting protein abundant in astrocytes and ependymal cells, that has been found to decrease osmotically-induced swelling. The purpose of this study was to examine whether PMA given at different time points following focal ischemia induced by middle cerebral artery occlusion (MCAO) reduces brain edema by AQP4 modulation. Male Sprague-Dawley rats were randomly assigned to sham procedure, vehicle, or PMA infusion (230 microg/kg), starting either 60 minutes before, or 30 or 60 minutes after MCAO (each group n = 12). After a 2-hour period of ischemia and 2 hours of reperfusion, the animals were sacrificed for assessment of brain water content, sodium, and potassium concentrations. AQP4 expression was assessed by immunoblotting. Statistical analysis was performed by ANOVA followed by Tukey's post hoc test. PMA treatment significantly reduced brain water content concentration in the infarcted area when started before or 30 minutes post-occlusion (p < 0.001, p = 0.022) and prevented the subsequent sodium shift (p < 0.05). Furthermore, PMA reduced ischemia-induced AQP4 up-regulation (p < 0.05). Attenuation of the ischemia-induced AQP4 up-regulation by PMA suggests that the reduction in brain edema formation following PMA treatment was at least in part mediated by AQP4 modulation.

Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury.

The effect of mild closed head trauma, induced by the weight-drop method (450 g from a 1-m height), on lipid peroxidation and energy metabolism of brain tissue was determined at various times after cerebral injury in spontaneously breathing rats (1, 10, 30 minutes and 2, 6, 15, 24, 48, and 120 hours). Animals were continuously monitored for the evaluation of blood pressure, blood gases, heart rate, and intracranial pressure. Analysis of malondialdehyde (MDA) as an index of lipid peroxidation, ascorbic acid, high-energy phosphates, nicotinic coenzymes, oxypurines, and nucleosides was performed by high-performance liquid chromatography (HPLC) on neutralized perchloric acid extract of the whole brain. Data showed that MDA, undetectable in control, sham-operated rats, was already present within 1 minute of trauma (1.77 nmol/g wet weight; SD = 0.29) and reached maximal values by 2 hours (72.26 nmol/g w.w.; SD = 11.26), showing a progressive slow decrease thereafter. In contrast, ATP, GTP, and nicotinic coenzyme (NAD and NADP) concentrations showed significant reduction only by the second hour postinjury. Maximal decrease of the ATP and GTP concentrations were seen at 6 hours postinjury, whereas NAD and NADP concentrations showed maximum decline by 15 hours. Values recorded in mechanically ventilated rats did not differ significantly from those obtained in spontaneously breathing animals. These findings, supported by the absence of blood gas and blood pressure changes in the spontaneously breathing rats, strongly support the premise that biochemical changes (primarily lipid peroxidation) are not caused by secondary ischemic-hypoxic phenomena but rather are triggered by these forces acting on the brain at the time of impact. In addition, these results suggest that depression of energy metabolism might be caused by peroxidation of the mitochondrial membrane with a consequent alteration of the main mitochondrial function-that is, the energy supply.

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity.

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe2+-H2O2 or Fe3+-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)+, only Fe2+-H2O2 also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe2+ iron (concentration range 3-180 microM) or H2O2 (concentration range 50-1000 microM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)+, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)+/ADP-ribose(P) ratio was about 4 at any Fe2+ or H2O2 concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)+/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)+ production of Fe2+-H2O2-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where "conventional" oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.

Activity and mechanism of the antioxidant properties of cyanidin-3-O-beta-glucopyranoside.

In the present study, the antioxidant activity, the interaction with reactive oxygen species and the redox potential of cyanidin-3-O-beta-glucopyranoside (C-3-G), the main anthocyanin present in juice of pigmented oranges, were evaluated in detail. C-3-G effects on low density lipoproteins (LDL) oxidation induced by 40 microM Cu at a pH of 7.4 were compared with those of resveratrol and ascorbic acid, two other natural antioxidants. All cyanidin-3-O-beta-glucopyranoside concentrations used (1, 2, 5, 10, 20, 50, 100 and 200 microM) inhibited malondialdehyde (MDA) generation (an index of lipid peroxidation), the inhibition being significantly higher than that obtained with equal concentrations of resveratrol and ascorbic acid (IC50 = 6.5 microM for C-3-G, 34 microM for resveratrol and 212 microM for ascorbic acid). Experiments of LDL oxidation performed at a pH of 5.0 or 6.0 showed that C-3-G antioxidant activity is not influenced by pH variations between 5.0 and 7.4. This suggests that metal chelation, exerted by C-3-G through the eventual dissociation of its phenolic groups, plays a minor role in its protective mechanism. The presence of C-3-G produced significantly higher protective effects of pigmented orange juice (obtained from Moro cultivar) with respect to blond orange juice, when tested on copper-induced LDL oxidation. The evaluation of the direct interaction with reactive oxygen species (H2O2, -O2, OH*), demonstrated that C-3-G is quickly oxidized by these compounds and it is, therefore, a highly efficient oxygen free radical scavenger. The powerful C-3-G antioxidant activity is in excellent agreement with the very negative redox potential (-405 mV), determined through direct current cyclic voltammetry measurements. On the basis of these results, C-3-G should be considered as one of the most effective antioxidants that can be assumed with dietary plants; therefore, pigmented oranges represent a very relevant C-3-G source because of the high content of this anthocyanin in their juice.

Early onset of lipid peroxidation after human traumatic brain injury: a fatal limitation for the free radical scavenger pharmacological therapy?

BACKGROUND: On the basis of the contradiction between data on experimental head trauma showing oxidative stress-mediated cerebral tissue damage and failure of the majority of clinical trials using free radical scavenger drugs, we monitored the time-course changes of malondialdehyde (MDA, an index of cell lipid peroxidation), ascorbate, and dephosphorylated ATP catabolites in cerebrospinal fluid (CSF) of traumatic brain-injured patients. METHODS: CSF samples were obtained from 20 consecutive patients suffering from severe brain injury. All patients were comatose, with a Glasgow Coma Scale on admission of 6 +/- 1. The first CSF sample for each patient was collected within a mean value of 2.95 hours from trauma (SD=1.98), after the insertion of a ventriculostomy catheter for the continuous monitoring of intracranial pressure. During the next 48 hours, CSF was withdrawn from each patient once every 6 hours. All samples were analyzed by an ion-pairing high-performance liquid chromatographic method for the simultaneous determination of MDA, ascorbic acid, hypoxanthine, xanthine, uric acid, inosine, and adenosine. RESULTS: In comparison with values recorded in 10 herniated-lumbar-disk, noncerebral control patients, data showed that all CSF samples of brain-injured patients had high values (0.226 micromol/L; SD=0.196) of MDA (undetectable in samples of control patients) and decreased ascorbate levels (96.25 micromol/L; SD=31.74), already at the time of first withdrawal at the time of hospital admission. MDA was almost constant in the next two withdrawals and tended to decrease thereafter, although 48 hours after hospital admission, a mean level of 0.072 micromol/L CSF (SD=0.026) was still recorded. The ascorbate level was normalized 42 hours after hospital admission. Changes in the CSF values of ATP degradation products (oxypurines and nucleosides) suggested a dramatic alteration of neuronal energy metabolism after traumatic brain injury. CONCLUSIONS: On the whole, these data demonstrate the early onset of oxygen radical-mediated oxidative stress, proposing a valid explanation for the failure of clinical trials based on the administration of oxygen free radical scavenger drugs and suggesting a possible rationale for testing the efficacy of lipid peroxidation "chain breakers" in future clinical trials.

Modulation of aquaporin-4 water transport in a model of TBI.

Our Laboratory has pursued the hypothesis that traumatic brain edema is predominantly cellular and recent supportive evidence has been obtained indicating a non-extracellular route for sodium and water entering brain. The aim of this study was to investigate if astrocytic endfeet are involved in this passage, using a potent activator of Protein Kinase C (phorbol ester) to modify and closing the Aquaporin 4 (AQP4), a water channel specific for astrocytic endfoot. Anaesthetized Sprague-Dawley rats were subjected to an intracerebroventricular bolus of phorbol ester (50 pmol/4 microl) or vehicle, in the right hemisphere and after 30 minutes they were exposed to the well-established conical contusion model (3 mm depth at 6 m/sec) on the same side. After trauma, they were subjected to 5 hours of drug continuous infusion, then sacrificed. Water content measurements for both right (injured) and left (uninjured) hemispheres were calculated using the wet weight/dry weight technique. Results of these experiments showed a significant decrease in water content in injured phorbol treated animals, underlying that AQP4 regulation plays an important role in brain edema following stroke, and supporting the concept of cellular formation for edema via astrocytic foot processes.

Exercise-induced oxidative stress in elderly subjects: the effect of red orange supplementation on the biochemical and cellular response to a single bout of intense physical activity.

Aging is characterized by an impaired capacity to maintain the redox balance both in physiological and pathological situations associated with an increased production of reactive oxygen species. Since the extent of this phenomenon may be influenced by an antioxidants-rich diet, we investigated the effect of supplementation with fresh red orange juice (ROJ) on biochemical and cellular biomarkers of oxidative stress in healthy, trained elderly women after a single bout of exhaustive exercise (EE). To this purpose, a sample of 22 females, 15 (69.0 ± 5.1 years) taking the ROJ supplementation and 7 (68.1 ± 2.7 years) as Control group, was constituted. Blood samples were collected immediately before, 30 minutes, and 24 hr after a single bout of EE, at baseline and after 4 weeks. Our results demonstrate that markers of DNA damage or apoptosis were not affected by EE both in Control and ROJ group, and by ROJ, whereas, exercise temporarily affected the redox balance in both groups. Controls didn't change their response to EE after the experimental period, but experimental group after ROJ supplementation had lower EE-induced MDA, consumed less ascorbic acid, and had less activation of the hypoxanthine/xanthine system, i.e., they seemed to be protected from hypoxia/reoxygenation mechanisms.

Modulation of peripheral markers of the serotoninergic system in healthy horses.

To evaluate the changes in plasma and platelet serotonin (5-HT) as markers of the serotoninergic system in equines, 5-HT content was measured by high performance liquid chromatography (HPLC) in deproteinized plasma obtained from peripheral blood samples of 12 clinically healthy horses, before and after feeding. 5-HT was measured in platelet rich plasma (PRP) and in platelet poor plasma (PPP). 5-HT in platelets (p5-HT) was obtained by subtracting free 5-HT in PPP (f5-HT) from 5-HT in PRP. After food ingestion, significant increases in p5-HT and f5-HT (p < 0.001), and no changes in the f5-HT/p5-HT ratio were recorded. Increase in the total circulating 5-HT might account both for initiating peristaltic activity and for increasing the f5-HT levels. Augmented 5-HT uptake by platelets could reflect the hypothetical increased activity of the serotoninergic neurons. Besides showing the feasibility to obtain f5-HT and p5-HT through HPLC determination of 5-HT in PRP and PPP equine plasma, these findings are consistent with the postulation that 5-HT is released from enterochromaffin cells following a mechanical and chemical stimulation.

Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress.

To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress.

Oxidative stress induces impairment of human erythrocyte energy metabolism through the oxygen radical-mediated direct activation of AMP-deaminase.

The effect of oxidative stress on human red blood cell AMP-deaminase activity was studied by incubating either fresh erythrocytes or hemolysates with H(2)O(2) (0.5, 1, 2, 4, 6, 8, and 10 mm) or NaNO(2) (1, 5, 10, 20, and 50 mm), for 15 min at 37 degrees C. AMP-deaminase tremendously increased by increasing H(2)O(2) or NaNO(2) at up to 4 and 20 mm, respectively (maximal effect for both oxidants was 9.5 and 6.5 times higher enzymatic activity than control erythrocytes or hemolysates, respectively). The incubation of hemolysates with iodoacetate (5-100 mm), N-ethylmaleimide (0.1-10 mm), or p-hydroxymercuribenzoate (0.1-5 mm) mimicked the effect of oxidative stress on AMP-deaminase, indicating that sulfhydryl group modification is involved in the enzyme activation. In comparison with control hemolysates, changes of the kinetic properties of AMP-deaminase (decrease of AMP concentration necessary for half-maximal activation, increase of V(max), modification of the curve shape of V(o) versus [S], Hill plots, and coefficients) were recorded with 4 mm H(2)O(2)- and 1 mm N-ethylmaleimide-treated hemolysates. Data obtained using 90% purified enzyme, incubated with Fenton reagents (Fe(2+) + H(2)O(2)) or -SH-modifying compounds, demonstrated that (i) reactive oxygen species are directly responsible for AMP-deaminase activation; (ii) this phenomenon occurs through sulfhydryl group modification; and (iii) the activation does not involve the loss of the tetrameric protein structure. Results of experiments conducted with glucose-6-phosphate dehydrogenase-deficient erythrocytes, challenged with increasing doses of the anti-malarial drug quinine hydrochloride and showing dramatic AMP-deaminase activation, suggest relevant physiopathological implications of this enzymatic activation in conditions of increased oxidative stress. To the best of our knowledge, this is the first example of an enzyme, fundamental for the maintenance of the correct red blood cell energy metabolism, that is activated (rather than inhibited) by the interaction with reactive oxygen species.

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts.

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.