Tm Bax Oxygen

Tm Bax Oxygen

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The finding of a dependence of oxygen consumption on oxygen delivery in critically ill patients has encouraged interventions to increase oxygen delivery index (DO2I) to overcome tissue hypoxia. In individuals other factors may influence oxygen consumption index (VO2I) and DO2I and may cause an apparently dependent relationship. We studied the effects of sedation and temperature on the VO2I/DO2I relationship in 13 perioperative patients. Pooled data showed significant correlations between VO2I and DO2I (r greater than 0.6, p less than 0.05) but also between VO2I and sedation score (r greater than 0.7, p less than 0.05), but not VO2I and temperature (r less than 0.5). When VO2I was standardized for the effects of sedation score (SS), the relationship between VO2I and DO2I was lost (r less than 0.5). Seven of 13 patients had significant (p less than 0.05) correlations between VO2I and SS and six of 13 between VO2I and DO2I; when standardized for the effect of varying sedation, no relationships were significant. When interpreting oxygen transport data from critically ill patients, the effects of sedation but not temperature must be taken into account; otherwise a false impression of a dependent relationship between VO2I and DO2I may cause unnecessary treatment.

This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

In the present study, we are the first to show data indicating that Lexa can significantly induce apoptosis in resistant HCC cell lines in the presence of cycloheximide (CHX). We provide evidence to demonstrate that treatment combining Lexa and CHX induces caspase-dependent apoptosis in HCC cells. Intracellular reactive oxygen species (ROS) generation, Bax/Bak activation, and heat shock protein 90 (HSP90) inactivation are involved in killing the HCC cells. More importantly, we found that Lexa and CHX combination treatment has no apparent apoptotic toxicity to normal human hepatocytes.

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

A major risk factor for pre-eclampsia is reduced uteroplacental blood flow in mid-pregnancy,6 suggesting that hypoxia could play an important role in the etiology of the disease. Several in vitro studies provide evidence that placental hypoxia is related to pathophysiology. For example, increased trophoblast shedding into the maternal circulation, which occurs in women with pre-eclampsia, is seen when villous tissues are cultured at 2% O2.7 Changes to the normal trophoblast life cycle occur under hypoxic conditions, evidenced both by increased rates of cytotrophoblast proliferation7 and reduced syncytial fusion in vitro.7, 8 Hypoxia (2% O2) also inhibits activin A production by trophoblast9 and downregulates syncytin expression,10 both of which are necessary for fusion. Alterations in the production of vasculogenic mediators such as vascular endothelial growth factor (VEGF), placental growth factor and soluble fms-like tyrosine kinase-1 (sFlt-1), from trophoblast cells occur under reduced oxygen,11, 12 similar to the alterations in production seen in pre-eclampsia, and these have also been proposed to contribute to disease pathogenesis.13, 14

A method for identifying pathogenic components in pre-eclampsia would aid our understanding of the disease and could also facilitate the identification of at-risk women. Preliminary characterization studies have demonstrated that the vasoactive factor in plasma is likely to be a protein or associated with a protein.15 Plasma complexity, and the fact that a few abundant proteins contribute to over 85% of its proteome,16 makes the direct identification of low-abundance proteins in maternal blood highly problematic. To test the hypothesis that oxygen regulates the production of soluble factors from the placenta, which influence vascular endothelial cell function, conditioned medium from explants exposed to different oxygen tensions was applied to endothelial cells in culture and bioassays measuring different aspects of endothelial cell function were conducted. The methodology described can form the basis of future protocols to identify pathogenic components of placental origin.

The effect of oxygen-conditioned explant medium on angiogenesis. (a) Light micrographs showing the appearance of tubules formed by UtMVEC seeded on Matrigel with either 20, 6 or 1% oxygen-conditioned explant medium. Tubule length (b) and tubule number (c) were determined by image analysis. Hypoxic explant medium (1% O2) caused both a significant reduction in average tubule length (b) (P

The release of vasoactive metabolites from cultured endothelial cells was assessed after incubation with explant-conditioned medium. Media under all oxygen concentrations stimulated UtMVEC to produce the vasodilator 6-keto-PGF1α over the 24-h incubation period (Figure 7a). Similarly, all media stimulated an equivalent rate of nitrite release from the endothelial cells (Figure 7b). The release of the vasoconstrictor ET-1 from cultured endothelial cells over the 24-h incubation period was also unaltered following exposure to hypoxic explant medium (Figure 7c). No statistically significant differences were therefore seen in the release of any vasoactive metabolite tested, following exposure of endothelial cells to explant medium from differing oxygen concentrations.

The effect of oxygen-conditioned placental medium on the release of vasoactive metabolites. The release of vasodilators 6-keto-PGF1α (a) and nitrite (b), and vasoconstrictor ET-1 (c), from cultured UtMVEC following the application of oxygen-conditioned placental explant medium. No significant changes in release were determined as the oxygen concentration decreased; N=8.

The measurement of liberated VEGF and sVEGFR-1 from the placental explants. Free VEGF and its soluble receptor sVEGFR-1 were measured in the explant-conditioned medium by immunoassay. Free VEGF was undetectable (not shown), but sVEGFR-1 was present in explant medium under all oxygen concentrations, with no significant changes in levels as the oxygen tension altered; N=8.

This study has demonstrated that hypoxia (1% O2) stimulates the liberation of factors from the placenta, which influence the metabolic status and the mitochondrial integrity of microvascular endothelial cells in vitro. Decreasing oxygen concentrations used during explant culture significantly altered the release of factors that affected viability, metabolism, angiogenic properties and cell death of primary cultured endothelial cells. These findings support a model of pre-eclampsia in which soluble factors released by the placenta cause the generalized endothelial pathology associated with the disease, and demonstrate the suitability of the explant model for studies of placenta-dependent pregnancy pathology.

Following exposure to explant-conditioned medium from each oxygen tension, a decreased rate of MTT reduction was seen when compared with cells in standard culture medium. Compared with medium under other oxygen tensions, the rate of MTT reduction was relatively lower following exposure of endothelial cells to hypoxic placental explant medium, indicating a greater alteration of oxidative metabolism and a decrease in the mitochondrial redox