The increased susceptibility to hydrogen peroxide of the (post)-ischemic rat heart is associated with the magnitude of the low molecular weight iron pool

Recently we have shown that intracellular low molecular weight (LMW) iron increases during ischemia. It is hypothesized that this increase in LMW iron during ischemia underlies the reported hydrogen peroxide toxicity toward ischemic hearts. To investigate this hypothesis, rat hearts were subjected to 15 min of no-flow ischemia and reperfused with buffer saturated against 95% N2 and 5% CO2 (anoxic reperfusuion) for 7 min. Hearts were then swithched to buffer saturated against 95% O2 and 5% CO2 (reoxygenation) to assess functional recovery. The cardiac function recovered to 80 ± 7% of the preischemic value. When the anoxic reperfusion was applied in the presence of 10 μM hydrogen peroxide, functional recovery after reoxygenation was 47 ± 7%. Hearts that were perfused with deferoxamine before ischemia and then subjected to ischemia and anoxic reperfusion in the presence of 10 μM hydrogen peroxide recovered to 78 ± 8%. Immediate reoxygenation after ischemia led to only 45 ± 6% recovery of function. During ischemia, LMW iron increased from 49 ± 45 to 183 ± 45 pmol/mg protein (p < .05) and decreasedto 58 ± 38 pmol/mg protein (p < .05) during the subsequent anoxic perfusion. Rat hearts preloaded with deferoxamine showed a slightly higher LMW iron content than normal (85 ± 23 and 49 ± 45 pmol/mg protein, respectively; n.s.), which showed a small, nonsignificant increase up to 136 ± 42 pmol/mg protein after 15 min of ischemia. No significant changes were found in reduced and oxidized glutathione content and glutathione peroxidase or catalase activities under those conditions. Our results indicate that hydrogen peroxide toxicity is determined by the amount of catalytic iron in the LMW pool and not by a decrease in antioxidant defense capacity to hydrogen peroxide

P38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level

The cytokine tumor necrosis factor (TNF) alpha was found to stimulate the p38 mitogen activated protein (MAP) kinase signalling cascade in human umbilical vein endothelial cells. TNFalpha increased the activity of the p38 substrate MAP kinase-activated-protein (MAPKAP) kinase 2 and the subsequent phosphorylation of the small heat shock protein Hsp27 about two to three fold. This stimulation was blocked almost completely by the specific p38 MAP kinase inhibitor SB203580. This inhibitor also suppressed the TNFalpha-induced surface expression of the endothelial adhesion molecule vascular cell adhesion molecule (VCAM)-1. In contrast, inhibition of p38 MAP kinase had no effect on the stimulated surface expression of the intercellular cell adhesion molecule (ICAM)-1. VCAM-1 mRNA accumulation induced by TNFalpha was not affected by SB203580, suggesting that the p38 MAP kinase signalling cascade regulates the endothelial expression of VCAM-1 at the post-transcriptional level

Involvement of reperfusion injury salvage Kinases in preconditioning depends critically on the preconditioning stimulus

Different preconditioning stimuli can activate divergent signaling pathways. In rats, adenosine-independent pathways (triple 3-min coronary artery occlusion [3CAO3]) and adenosine-dependent pathways (one 15-min coronar

The role of superoxide anions in the development of distant tumour recurrence

We hypothesise that reactive oxygen species (ROS) released from activated polymorphonuclear leucocytes during surgery play a crucial role in enhanced tumour recurrence seen after surgery. Therefore, the effect of ROS on adhesion of tumour cells to microvascular endothelium in a reproducible human in vitro model was studied. Preincubation of microvascular endothelial cells with the superoxide anion producing xanthine-xanthine oxidase complex significantly increased adhesion of the human colon carcinoma cells HT29 (167% vs control, P<0.01), Caco2 (164% vs control, P<0.01) and of the pancreas carcinoma cells PanC1 (180% vs control, P<0.01). Addition of the antioxidant enzymes superoxide dismutase or catalase significantly decreased tumour cell adhesion (P<0.01). Exposure of endothelial cells to superoxide anions increased the apoptotic rate to 7.9 times the normal rate. Additionally, exposure increased expression of the endothelial adhesion molecules E-Selectin, ICAM-1, and VCAM-1 of maximally 170% vs control (P<0.01). In conclusion, this study shows that superoxide anions promote the adherence of tumour cells to the microvasculature by inducing endothelial apoptosis that subsequently induces the expression of various adhesion molecules for tumour cells. This indicates that by tackling the production of ROS preventing tumour recurrence at distant sites might be feasible

Altered expression of mitochondrial electron transport chain proteins and improved myocardial energetic state during late ischemic preconditioning

Altered expression of mitochondrial electron transport proteins has been shown in early preconditioned myocardial tissue. We wished to determine whether these alterations persist in the Second Window of Protection (SWOP) and if so, whether a favorable energetic state is facilitated during subsequent ischemia. Fourteen pigs underwent a SWOP protocol with ten 2-minute balloon inflations in the LAD artery, each separated by 2 minutes reperfusion. Twenty-four hours later, mitochondria were isolated from SWOP and SHAM pig hearts and analyzed for uncoupling protein (UCP)-2 content by western blot analysis, proteomic changes by iTRAQ® and respiration by an oxygen electrode. In parallel in vivo studies, high-energy nucleotides were obtained by transmural biopsy from anesthetized SWOP and SHAM pigs at baseline and during sustained low-flow ischemia. Compared with SHAM mitochondria, ex vivo SWOP heart tissue demonstrated increased expression of UCP-2, Complex IV (cytochrome c oxidase) and Complex V (ATPase) proteins. In comparison with SHAM pigs during in vivo conditions, transmural energetics in SWOP hearts, as estimated by the free energy of ATP hydrolysis (ΔG 0), were similar at baseline but had decreased by the end of low-flow ischemia (-57.0 ± 2.1 versus -51.1 ± 1.4 kJ/mol; P < 0.05). In conclusion, within isolated mitochondria from preconditioned SWOP hearts, UCP-2 is increased and in concert with enhanced Complex IV and V proteins, imparts a favorable energetic state during low-flow ischemia. These data support the notion that mitochondrial adaptations that may reduce oxidant damage do not reduce the overall efficiency of energetics during sustained oxygen deprivation

Rapid ultraperformance liquid chromatography-tandem mass spectrometry assay for a characteristic glycogen-derived tetrasaccharide in pompe disease and other glycogen storage diseases

BACKGROUND: Urinary excretion of the tetrasaccharide 6-α-D- glucopyranosyl-maltotriose (Glc4) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc4 a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc4 in urine without interference of the Glc4 isomer maltotetraose (M4). METHODS: Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS. RESULTS: We separated and quantified acarbose, M4, and Glc4 using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc4 was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. In