The role of mitochondrial oxidation in endotoxin-induced liver-dependent swine pulmonary edema.

UNLABELLED: We reported previously studies in an in situ perfused swine preparation demonstrating that endotoxemia induced lung injury required the presence of the liver and that the response was accompanied by oxidative stress. To determine whether lung and liver mitochondrial oxidative stress was important to the response, we compared the effects of equimolar amounts of two antioxidants, n-acetylcysteine, which does not replenish mitochondrial glutathione, and procysteine which does, on endotoxemia induced lung injury in the swine preparation. In a swine perfused liver-lung preparation, we measured physiologic, biochemical and cellular responses of liver and lung to endotoxemia with and without the drugs. Endotoxemia caused oxidation of the mitochondria-specific protein, thioredoxin-2, in both the lungs and the liver. Procysteine reduced thioredoxin-2 oxidation, attenuated hemodynamic, gas exchange, hepatocellular dysfunction, and cytokine responses and prevented lung edema. n-acetylcysteine had more modest effects and did not prevent lung edema. CONCLUSIONS: We conclude that mitochondrial oxidation may be critical to the pathogenesis of endotoxemia-induced liver-dependent lung injury and that choices of antioxidant therapy for such conditions must consider the desired subcellular target in order to be optimally effective.

Potent effects of aerosol compared with intravenous treprostinil on the pulmonary circulation.

Inhaled vasodilator therapy for pulmonary hypertension may decrease the systemic side effects commonly observed with systemic administration. Inhaled medications only reach ventilated areas of the lung, so local vasodilation may improve ventilation-perfusion matching and oxygenation. We compared the effects of intravenous vs. aerosolized treprostinil on pulmonary and systemic hemodynamics in an unanesthetized sheep model of sustained acute pulmonary hypertension. Acute, stable pulmonary hypertension was induced in instrumented unanesthetized sheep by infusing a PGH(2) analog, U-44069. The sheep were then administered identical doses of treprostinil either intravenously or by aerosol. Systemic and pulmonary hemodynamics were recorded during each administration. Both intravenous and aerosol delivery of treprostinil reduced pulmonary vascular resistance and pulmonary arterial pressure, but the effect was significantly greater with aerosol delivery (P < 0.05). Aerosol delivery of treprostinil had minimal effects on systemic hemodynamics, whereas intravenous delivery increased heart rate and cardiac output and decreased left atrial pressure and systemic blood pressure. Aerosol delivery of the prostacyclin analog treprostinil has a greater vasodilatory effect in the lung with minimal alterations in systemic hemodynamics compared with intravenous delivery of the drug. We speculate that this may result from treprostinil stimulated production of vasodilatory mediators from pulmonary epithelium.

Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model.

INTRODUCTION: The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. METHODS: A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. RESULTS: Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1ß and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. CONCLUSIONS: We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic responses induced by endotoxemia by modulating the inflammatory response, mechanisms that do not involve engraftment or trans-differentiation of the cells. These observations may have important implications for the design of future cell therapies for ARDS.

High-performance metabolic profiling of plasma from seven mammalian species for simultaneous environmental chemical surveillance and bioeffect monitoring.

High-performance metabolic profiling (HPMP) by Fourier-transform mass spectrometry coupled to liquid chromatography gives relative quantification of thousands of chemicals in biologic samples but has had little development for use in toxicology research. In principle, the approach could be useful to detect complex metabolic response patterns to toxicologic exposures and to detect unusual abundances or patterns of potentially toxic chemicals. As an initial study to develop these possible uses, we applied HPMP and bioinformatics analysis to plasma of humans, rhesus macaques, marmosets, pigs, sheep, rats and mice to determine: (1) whether more chemicals are detected in humans living in a less controlled environment than captive species and (2) whether a subset of plasma chemicals with similar inter-species and intra-species variation could be identified for use in comparative toxicology. Results show that the number of chemicals detected was similar in humans (3221) and other species (range 2537-3373). Metabolite patterns were most similar within species and separated samples according to family and order. A total of 1485 chemicals were common to all species; 37% of these matched chemicals in human metabolomic databases and included chemicals in 137 out of 146 human metabolic pathways. Probability-based modularity clustering separated 644 chemicals, including many endogenous metabolites, with inter-species variation similar to intra-species variation. The remaining chemicals had greater inter-species variation and included environmental chemicals as well as GSH and methionine. Together, the data suggest that HPMP provides a platform that can be useful within human populations and controlled animal studies to simultaneously evaluate environmental exposures and biological responses to such exposures.

Endotoxin-induced acute lung injury requires interaction with the liver.

Clinical and laboratory data indicate that the liver plays an important role in the incidence, pathogenesis, and outcome of acute lung injury/acute respiratory distress syndrome. To distinguish direct effects of endotoxin on the lungs from liver-dependent effects during the early phase of the response to endotoxemia, we used an in situ perfused piglet preparation in which only the ventilated lung or both the lung and liver could be included in a blood perfused circuit. We monitored pulmonary vascular resistance, oxygenation, neutrophil count, lung edema as reflected by wet-dry weights of lung tissue, perfusate concentrations of TNF-alpha, IL-6, and 8-isoprostane (a marker of oxidative stress), and activation of the transcription factor (NF-kappaB) in lung tissue before and for 2 h after endotoxin. When only the lung was perfused, endotoxin caused pulmonary hypertension and neutropenia; but oxygenation was maintained; TNF-alpha, IL-6, and 8-isoprostane levels were minimally elevated; and there was no lung edema. When both the liver and lung were perfused, endotoxin caused marked hypoxemia, large increases in perfusate TNF-alpha, IL-6, and 8-isoprostane concentrations, and severe lung edema. NF-kappaB activation in the lung was greatest when the liver was in the perfusion circuit. We conclude that the direct effects of endotoxemia on the lungs include vasoconstriction and leukocyte sequestration, but not lung injury. Intense activation of the inflammatory response and oxidative injury that results in pulmonary edema and hypoxemia (acute lung injury) requires interaction of the lungs with the liver.

ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery.

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ET(B) in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.

Comparing transforming growth factor-beta2, talc and bleomycin as pleurodesing agents in sheep.

BACKGROUND: Transforming growth factor (TGF)-beta2 can produce effective pleurodesis in animals, but its efficacy has not been compared with commonly used pleurodesing agents in sheep, which have a thick pleura resembling that of humans. The acute physiological effects and the level of systemic TGF-beta absorption after its intrapleural administration have not been studied. The aims of the present study were to compare: (i) the effectiveness of TGF-beta2, talc and bleomycin in producing pleurodesis in sheep; (ii) the acute side-effects and systemic TGF-beta levels following the intrapleural administration of these agents; and (iii) histological changes after intrapleural injections of these agents. METHODOLOGY: Twelve sheep were divided into three groups and were given a single intrapleural dose of TGF-beta2 (0.25 microg/kg), talc slurry (5 g) or bleomycin (60 IU) via a chest tube. Saline or buffer was injected into the contralateral side, which served as the control. Arterial blood gases and respiratory and heart rates were monitored for the first 24 h. Plasma levels of TGF-beta1 and TGF-beta2 were measured. Pleurodesis was graded macroscopically from 1 (none) to 8 (symphysis > 50% of hemithorax) at day 14. RESULTS: At day 14, the pleurodesis score of the TGF-beta2 group (7.7+/-0.6) was similar to that of the talc (7.0+/-1.7) group and significantly higher than that of the bleomycin group (3.3+/-2.3; P < 0.05). No significant differences were seen in arterial blood gas analysis, vital signs and plasma TGF-beta1 and TGF-beta2 concentrations among the three groups. CONCLUSIONS: Transforming growth factor-beta2 was as effective as talc and more so than bleomycin in inducing pleurodesis in sheep. Intrapleural administration of TGF-beta2 appeared safe. No acute changes in gaseous exchange or macroscopic abnormalities were seen following intrapleural TGF-beta2. Importantly, there was no evidence of an increase in systemic TGF-beta levels following its intrapleural administration.