Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

Proliferation of C6 glioma cells requires the phospholipid remodeling enzyme tafazzin independent of cardiolipin composition.

The mitochondrial phospholipid (CL) has been linked to mitochondrial and cellular functions. It has been postulated that the composition of CL is of impact for mitochondrial energy metabolism and cell proliferation. Although a correlation between CL composition and proliferation could be demonstrated for several cell types, evidence for a causal relationship remains obscure. Here, we applied two independent approaches, i) supplementation of fatty acids and ii) knock-out of the phospholipid remodeling enzyme tafazzin, to manipulate CL composition and analyzed the response on proliferation of C6 glioma cells. Both strategies caused substantial changes in the distribution of cellular fatty acids as well as in the distribution of fatty acids incorporated in CL that were accompanied by changes of the composition of molecular CL species. These changes did not correlate with cell proliferation. However, knock-out of tafazzin caused dramatic reduction in proliferation of C6 glioma cells independent of CL composition. The mechanism of tafazzin-dependent restriction of proliferation remains unclear. Among the various fatty acids administered only palmitic acid restricted cell proliferation by induction of cell death.

Activated clotting factor X mediates mitochondrial alterations and inflammatory responses via protease-activated receptor signaling in alveolar epithelial cells.

There is growing evidence for the contribution of the activated coagulation factor X (FXa) in the development of chronic inflammatory lung diseases. Therefore, we aimed to investigate effects of exogenous FXa on mitochondrial and metabolic function as well as the induction of inflammatory molecules in type II alveolar epithelial cells. Effects of FXa on epithelial cells were investigated in A549 cell line. Activation of extracellular signal-regulated kinase (ERK) and induction of inflammatory molecules were examined by immunoblot and gene expression analysis. Mitochondrial function was assessed by the measurement of oxygen consumption during maximal oxidative phosphorylation and quantitative determination of cardiolipin oxidation. Apoptosis was tested using a caspase 3 antibody. Metabolic activity and lactate dehydrogenase assay were applied for the detection of cellular viability. FXa activated ERK1/2 and induced an increase in the expression of pro-inflammatory cytokines, which was prevented by an inhibitor of FXa, edoxaban, or an inhibitor of protease-activated receptor 1, vorapaxar. Exposure to FXa caused mitochondrial alteration with restricted capacity for ATP generation, which was effectively prevented by edoxaban, vorapaxar and GB83 (inhibitor of protease-activated receptor 2). Of note, exposure to FXa did not initiate apoptosis in epithelial cells. FXa-dependent pro-inflammatory state and impairment of mitochondria did not reach the level of significance in lung epithelial cells. However, these effects might limit regenerative potency of lung epithelial cells, particular under clinical circumstances where lung injury causes exposure to clotting factors.

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions, and cellular proliferation.

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.

Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells.

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72 h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.

Lipidomic analysis of molecular cardiolipin species in livers exposed to ischemia/reperfusion.

Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.

Oxidation of cardiolipin is involved in functional impairment and disintegration of liver mitochondria by hypoxia/reoxygenation in the presence of increased Ca²âº concentrations.

The aim of this study was to investigate the interrelationship between the mitochondrial phospholipid cardiolipin (CL), mitochondrial respiration and morphology in dependence on hypoxia/reoxygenation and Ca(2+). Therefore, we subjected rat liver mitochondria to hypoxia/reoxygenation at different extramitochondrial Ca(2+) concentrations and analysed mitochondrial respiration, morphology, CL content, the composition of molecular CL species, oxidation of CL and two mono-lyso-CL species. Hypoxia/reoxygenation in the presence of elevated extramitochondrial Ca(2+) concentration caused dramatic impairment of mitochondrial respiration and morphology. Concomitantly, increased amounts of oxidised CL were detected in the incubation medium after the treatment. Hypoxia/reoxygenation alone caused degradation of CL. The treatments had no effect on the composition of molecular CL species. Our data support the hypothesis that CL oxidation and CL degradation are involved in mitochondrial injury caused by hypoxia/reoxygenation and Ca(2+). Our results further suggest that prevention of CL oxidation by modification of CL composition may support the beneficial action of antioxidants during hypoxia/reoxygenation in the presence of elevated Ca(2+) concentrations.

Composition of molecular cardiolipin species correlates with proliferation of lymphocytes.

The mitochondrial phospholipid cardiolipin (CL) is required for oxidative phosphorylation. Oxidation of CL results in the disruption of CL-cytochrome c binding and the induction of apoptosis. Large variations in the acyl-chain residues of CL have been reported, but evidence as to whether these variants exert distinct biological effects has been limited. We have studied the acyl-chain composition of CL in lymphocytes, and found marked differences between highly and slowly proliferating cells. In fast growing cells, we detected a decreased number of double bonds, and a higher amount of C16 acyl-chain residues in CL, compared with slower growing cells. However, fewer C18 acyl-chain residues were found in CL from fast growing cells compared with slower proliferating cells. Our results suggest a functional link between acyl-chain composition of CL and cell proliferation.

Nrf2-dependent gene expression is affected by the proatherogenic apoE4 genotype-studies in targeted gene replacement mice.

An apoE4 genotype is an important risk factor for cardiovascular and other chronic diseases. The higher cardiovascular disease risk of apoE4 carriers as compared to the apoE3 genotype has been mainly attributed to the differences in blood lipids between the two genotype subgroups. Recently, a potential protective role of the transcription factor Nrf2 in cardiovascular disease prevention has been suggested. In this study, we show that Nrf2-dependent gene expression is affected by the apoE genotype. ApoE4 vs. apoE3 mice exhibited lower hepatic Nrf2 nuclear protein levels. Furthermore, mRNA and protein levels of Nrf2 target genes including glutathione-S-transferase, heme oxygenase-1 and NAD(P)H dehydrogenase, quinone 1 were significantly lower in apoE4 as compared to apoE3 mice. Lower hepatic mRNA levels of phase II enzymes, as observed in apoE4 vs. apoE3 mice, were accompanied by higher mRNA levels of phase I enzymes including Cyp26a1 and Cyp3a16. Furthermore, miRNA-144, miRNA-125b, and miRNA-29a involved in Nrf2 signaling, inflammation, and regulation of phase I enzyme gene expression were affected by the apoE genotype. We provide first evidence that Nrf2 is differentially regulated in response to the apoE genotype.

Palmitate protects hepatocytes from oxidative stress and triacylglyceride accumulation by stimulation of nitric oxide synthesis in the presence of high glucose and insulin concentration.

Excessive flux of free fatty acids (FFA) into the liver contributes to liver impairment in non-alcoholic fatty liver disease (NAFLD). It remains unclear how FFA contribute to impairment of hepatocytes. This study treated hepatocytes with linoleic acid and palmitate to investigate the early event triggering FFA-mediated impairment. It determined cell viability, content of nitrite/nitrate and triacylglycerides (TG), inducible nitric oxide synthase (iNOS) protein, oxidation of cardiolipin (CL) as well as formation of F(2)-isoprostanes in the presence of insulin and glucose. Linoleic acid caused significant decrease in cell viability. It is shown that palmitate caused induction of iNOS resulting in increased nitrite/nitrate concentration and slight increase in TG content. Linoleic acid led to a decrease in nitrite/nitrate concentration parallelled by massive TG accumulation in combination with increased oxidation of CL and increased F(2)-isoprostane levels. It is concluded that nitric oxide (NO) concentration regulates FFA-dependent TG accumulation and oxidative stress in rat hepatocytes.

Degradation of phospholipids by oxidative stress--exceptional significance of cardiolipin.

The aim of this study was to investigate the effect of oxidative stress on mitochondrial phospholipids. In this context, this study investigated (i) the content of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cardiolipin (CL), (ii) the correlation of CL degradation with mitochondrial function and (iii) the correlation of CL degradation and CL oxidation. Oxidative stress induced by iron/ascorbate caused a dramatic decrease of these phospholipids, in which CL was the most sensitive phospholipid. Even moderate oxidative stress by hypoxia/reoxygenation caused a decrease in CL that was parallelled by a decrease in active respiration of isolated rat heart mitochondria. The relation between oxidative stress, CL degradation and CL oxidation was studied by in vitro treatment of commercially available CL with superoxide anion radicals and H2O2. The degradation of CL was mediated by H2O2 and required the presence of cytochrome c. Other peroxidases such as horse radish peroxidase and glutathione peroxidase had no effect. Cytochrome c in the presence of H2O2 caused CL oxidation. The data demonstrate that oxidative stress may cause degradation of phospholipids by oxidation, in particular CL; resulting in mitochondrial dysfunction.

Inverse correlation of protein oxidation and proteasome activity in liver and lung.

Several studies have demonstrated that proteasome activity decreases whereas protein oxidation increases with aging in various tissues. However, no studies are available correlating both parameters directly comparing different tissues of one organism. Therefore, we determined whether there is an age-related change in proteasome activity and protein oxidation in heart, lung, liver, kidney and skeletal muscle samples of 6-, 10-, 18- and 26-month-old rats. There was a significant age-related increase in protein carbonyls at 18 and 26 months compared to young rats. Thereby, protein carbonyl formation was rather due to a general than a specific protein carbonylation as shown by immunblot studies. The highest increase in protein carbonyl formation was found in liver, lung and kidney samples. Proteasome activity decreased significantly with age in lung and liver samples. Proteasome activity in liver and lung decreased by factor five compared to young rats. Strong correlations between proteasome activity and protein oxidation were found in liver and lung, whereas in other tissues only a trend was found. These results demonstrate that the increase in protein oxidation and the decline in proteasome activity are correlating. Further studies are needed to determine the mechanisms which cause organ-specific aging-rates and their consequences.

Dietary vitamin E, brain redox status and expression of Alzheimer's disease-relevant genes in rats.

Oxidative stress is one of the major pathological features of Alzheimer's disease (AD). Here, we investigated whether dietary vitamin E (VE) depletion may induce adverse effects and supplementation with alpha-tocopherol (alphaT) may result in beneficial effects on redox status and the regulation of genes relevant in the pathogenesis of AD in healthy rats. Three groups of eight male rats each were fed diets with deficient ( < 1 mg alphaT equivalents/kg diet), marginal (9 mg alphaT equivalents/kg diet) or sufficient (18 mg alphaT equivalents/kg diet) concentrations of natural-source VE for 6 months; a fourth group was fed the VE-sufficient diet fortified with alphaT (total VE, 146 mg alphaT equivalents/kg diet). Feeding of the experimental diets dose dependently altered alphaT concentrations in the cortex and plasma. No significant changes in F2-isoprostane concentrations, activities of antioxidative enzymes (total superoxide dismutase, Se-dependent glutathione peroxidase) and concentrations of glutathione or the expression of AD-relevant genes were observed. In this non-AD model, depletion of VE did not induce adverse effects and supplementation of alphaT did not induce positive effects on the parameters related to the progression of AD.

Acidic peptides enhanced genistein-dependent inhibition of human platelet aggregation: potential protective effect of digestible peptides plus genistein against atherosclerosis.

The leading cause of death in the United States and European countries is coronary heart disease. We hypothesized that the ingestion of soy compounds may not only have beneficial effects on atherosclerotic risk by lowering lipid compounds, but also by reducing platelet aggregability. Therefore, we analyzed in vitro the influence of defined and digestible peptides, frequently found in glycinin and beta-conglycinin as important proteins of soy bean, on platelet aggregation of 180 healthy volunteers with or without the isoflavone genistein by aggregometry and flow cytometry. (i) The predominating share of amino acids and acidic, neutral, and basic di- and tripeptides of up to 2 mmol/L did not modify platelet aggregation induced by collagen, adenosine diphosphate, epinephrine, or arachidonic acid. (ii) Genistein inhibited agonist-induced platelet aggregation dose dependently. (iii) In the presence of the acidic peptides glutamate-glutamate and aspartate-aspartate-aspartate (1 mmol/L each), genistein reduced collagen- and ADP-dependent platelet activation stronger than 250 micromol/L of this isoflavone alone. Other peptides were less effective (eg, glutamate-glutamate-glutamate) or ineffective (eg, asparagine-asparagine). (iv) Glutamate-glutamate-glutamate (1 nmol/L), glutamate-glutamate (1 micromol/L), and aspartate-aspartate-aspartate (1 micromol/L) enhanced the inhibition of genistein on platelet aggregation induced by arachidonic acid. Thus, the results of the present in vitro investigation allow the assumption that nutrition with specific compounds of soy--acidic peptides together with genistein--might protect against coronary atherosclerosis by attenuating platelet activity. In vivo studies are warranted to check this assumption.

Mitochondrial dysfunction and redox signaling in atrial tachyarrhythmia.

Accumulating evidence links calcium-overload and oxidative stress to atrial remodeling during atrial fibrillation (AF). Furthermore, atrial remodeling appears to increase atrial thrombogeneity, characterized by increased expression of adhesion molecules. The aim of this study was to assess mitochondrial dysfunction and oxidative stress-activated signal transduction (nuclear factor-kappaB [NF-kappa B], lectin-like oxidized low-density lipoprotein receptor [LOX-1], intercellular adhesion molecule-1 [ICAM-1], and hemeoxgenase-1 [HO-1]) in atrial tissue during AF. Ex vivo atrial tissue from patients with and without AF and, additionally, rapid pacing of human atrial tissue slices were used to study mitochondrial structure by electron microscopy and mitochondrial respiration. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunoblot analyses, gel-shift assays, and enzyme-linked immunosorbent assay (ELISA) were applied to measure nuclear amounts of NF-kappa B target gene expression. Using ex vivo atrial tissue samples from patients with AF we demonstrated oxidative stress and impaired mitochondrial structure and respiration, which was accompanied by nuclear accumulation of NF-kappa B and elevated expression levels of the adhesion molecule ICAM-1 and the oxidative stress-induced markers HO-1 and LOX-1. All these changes were reproduced by rapid pacing for 24 hours of human atrial tissue slices. Furthermore, the blockade of calcium inward current with verapamil effectively prevented both the mitochondrial changes and the activation of NF-kappa B signaling and target gene expression. The latter appeared also diminished by the antioxidants apocynin and resveratrol (an inhibitor of NF-kappa B), or the angiotensin II receptor type 1 antagonist, olmesartan. This study demonstrates that calcium inward current via L-type calcium channels contributes to oxidative stress and increased expression of oxidative stress markers and adhesion molecules during cardiac tachyarrhythmia.

Impairment of endothelial nitric oxide synthase causes abnormal fat and glycogen deposition in liver.

Nitric oxide (NO) affects fatty acid synthesis and biogenesis of fatty acid consuming mitochondria. However, whether NO generated by the endothelial NO synthase isoform (eNOS) has significant impact on the synthesis and deposition of fat in liver remained unclear. We analyzed the quantity and distribution of mitochondria and fat in liver of wild-type (WT) mice and mice lacking eNOS (eNOS-KO). The livers of eNOS-KO mice contained tenfold more fat close (zone 1) and twenty fold more distal (zone 3) to the artery. The fat was deposited as droplets co-localized with mitochondria. Additionally, the livers of eNOS-KO mice contained 1.5-fold more homogenously distributed glycogen. No difference in the quantity of mitochondria was found between liver homogenates of eNOS-KO mice and WT animals. Mitochondria from liver homogenates of eNOS-KO mice exhibited a higher ratio of citrate synthase (CS) and NADH-cytochrome c oxidoreductase (KI+III) activity. We conclude that lack of eNOS-derived NO stimulates citrate- and lipid synthesis in liver thus contributing to the development of overweight. In support of this view, more visceral fat and 70% higher body weight was determined in one year old eNOS-KO mice in comparison to WT animals.

Serum Concentrations of F2-Isoprostanes and 4-Hydroxynonenal in Hemodialysis Patients in Relation to Inflammation and Renal Anemia.

BACKGROUND: Patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD) are apparently exposed to enhanced oxidative stress and to inflammation. It was the aim of this study to characterize the state of systemic oxidative stress of ESRD patients before and following HD using highly specific biomarkers, F(2)-isoprostanes and 4-hydroxynonenal (HNE). Furthermore the question should be answered, if there are associations between inflammation and systemic oxidative stress and/or between systemic oxidative stress and renal anemia, which is more or less typical for HD patients. PATIENTS AND METHODS: Concentrations of F(2)-isoprostanes, HNE, C-reactive protein (CRP) as marker of inflammation, and hemoglobin were measured in serum samples of patients with ESRD before and after HD and of healthy control persons for comparison. Total (esterified plus free) F(2)-isoprostanes were quantified by highly sensitive gas chromatography/mass spectrometry technique, HNE by thin layer chromatography and HPLC/UV detection, CRP by immunoturbidimetry and hemoglobin by clinico-chemical routine assay. RESULTS: 1. HD patients showed significantly higher serum concentrations of F(2)-isoprostanes and HNE than healthy human control subjects. 2. Total (esterified plus free) F(2)-isoprostane levels before HD were not significantly different from those after HD, whereas HNE levels were significantly decreased in patients after HD. 3. F(2)-isoprostane concentrations in HD patients correlated with the levels of CRP, whereas HNE concentrations inversely correlated with the content of hemoglobin. CONCLUSION: Both, F(2)-isoprostanes and HNE serum concentrations are useful oxidative stress parameters in ESRD patients undergoing HD. Whereas HNE strongly correlates with the severity of renal anemia, leading to left heart insufficiency, F(2)-isoprostanes (sum of free plus esterified) highly correlate with the degree of inflammation.

UVB irradiation-induced impairment of keratinocytes and adaptive responses to oxidative stress.

UVB irradiation of human skin is known to induce pathophysiological processes as oxidative stress and inflammation. HaCaT keratinocytes represent a well-established in vitro model system to investigate the influence of UVB irradiation on cell cultures. It was the aim of these investigations to study the effects of moderate UVB doses on cellular and mitochondrial integrity of HaCaT keratinocytes, biomarkers of oxidative stress and antioxidant protection by superoxide dismutases. F(2)-isoprostane concentrations were UVB dose-dependently enhanced reaching a plateau at 50 mJ/cm(2). Cell viability was reduced and apoptosis was enhanced with increasing UVB doses. The activities of the respiratory chain complexes were practically not altered at lower UVB doses, up to 50 mJ/cm(2), whereas remarkable decreases, also for the levels of cardiolipin species, were seen at 100 mJ/cm(2). As an adaptive response to the enhanced oxidative stress, protein levels of MnSOD increased about 3-fold at 50 mJ/cm(2) and decreased at higher doses. From the data it can be concluded that keratinocytes are sufficiently protected at low UVB doses, whereas higher doses lead to irreversible cell damage.