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Endothelial cell (EC) injury is observed in clinically important pathological processes, including bacterial endotoxemia. We hypothesized that such pathological processes may exhibit target organ heterogeneity due to organ-specific heterogeneity of endothelial cells. To test this hypothesis, endothelial cells of aorta (AO), pulmonary artery (PA), left ventricle (LV), and right ventricle (RV) were cultured from individual sheep and exposed to bacterial endotoxin. Marked heterogeneity in endotoxin-induced cytotoxicity was observed. AOEC were the most sensitive, followed by PAEC, LVEC, and RVEC. This cytotoxicity was manifested as programmed cell death (apoptosis). All cells were able to express both interleukin-6 and endothelin-1 (ET-1) transcripts. Following exposure to bacterial endotoxin, interleukin-6 transcripts accumulated in all cells, whereas ET-1 expression was constant or slightly decreased. These data suggest that organ-specific heterogeneity of EC responsiveness to endotoxin is a potential determinant of organ-specific resistance to endotoxin and other mediators of injury.

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We previously reported that sequential exposure of cultured porcine aortic endothelial cells to lipopolysaccharide (LPS) and then to inducers of the heat shock response resulted in lethal injury by programmed cell death. In these present experiments, we evaluated the ability of other mediators of sepsis to prime endothelial cells for subsequent injury upon heat shock induction. We used SVEC4-10 microvascular endothelial cells, a cloned SV40-transformed cell line derived from LPS-resistant C3H/HeJ mice. When these endothelial cells were treated first with tumor necrosis factor-alpha followed by induction of the heat shock response with either heat or sodium arsenite (As), a standard chemical inducer of the heat shock response in vitro, a tumor necrosis factor dose-dependent cytotoxicity was observed, similar to that which we had seen previously in porcine endothelial cells primed with LPS. Subsequent experiments found that priming with interferon-gamma produced a similar dose-dependent toxicity upon heat shock with either heat or As. The reducing agents dithiothreitol and n-acetylcysteine, which we have previously shown to inhibit heat shock-induced programmed cell death in LPS-primed porcine endothelial cells, were also effective in protecting against heat shock-induced death following cytokine-priming in this microvascular cell line, suggesting that the intracellular signaling pathways of these priming agents are somewhere convergent. These data suggest that both exogenous and endogenous mediators of inflammation can prime endothelial cells for subsequent injury upon induction of the heat shock response, and are consistent with the emerging concept of redundancy in inflammatory signaling.

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When cultured porcine endothelial cells are exposed first to endotoxin (lipopolysaccharide (LPS)) followed by standard inducers of the heat shock response in vitro (heat or sodium arsenite), these cells aberrantly execute programmed cell death. This cell death is dependent upon two distinct events: the LPS-priming step and the heat shock-induced activation step. Prior work demonstrated that the LPS-priming step could be blocked by the prior application of cell-permeable hydroxyl radical scavengers, suggesting a role for this reactive oxygen species as an important intracellular signal mediating the first step. In these present experiments, we evaluated the potential role of reduction-oxidation mechanisms in the heat shock activation step. The thiol reducing agents reduced glutathione (GSH), n-acetylcysteine (NAC), and dithiothreitol (DTT) were evaluated for their ability to block programmed cell death in LPS-primed porcine aortic endothelial cells. Both DTT and NAC, agents that augment intracellular reduced glutathione levels, were protective against cell death when applied prior to heat shock induction with sodium arsenite (As) in endothelial cells treated previously with LPS. The less cell permeable agent GSH was not protective. Delayed application of DTT or NAC could block progression to cell death for up to 1.5 h after initiation of the heat shock response with As. These data show that heat shock-induced programmed cell death in LPS-primed endothelial cells can be arrested, at least in its early stages, by agents that augment or stabilize the reducing potential of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

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OBJECTIVE: To evaluate the potential role of reactive oxygen metabolites as signals for endothelial cell apoptosis. DESIGN: A series of antioxidants were evaluated for their ability to block apoptosis in cultured porcine aortic endothelial cells in vitro. RESULTS: Scavenging of the hydroxyl radical with the membrane-permeable scavenger dimethyl sulfoxide or blocking its generation via the Fenton reaction by the chelation of iron with o-phenanthroline blocked apoptosis, whereas the cell membrane-impermeable scavengers superoxide dismutase and catalase did not block apoptosis. Inhibition of xanthine oxidase with enzyme-inhibitory levels of allopurinol also failed to block apoptosis, whereas high levels of allopurinol, which also scavenge the hydroxyl radical in vitro, conferred protection. In each case (dimethyl sulfoxide, o-phenanthroline, and high-dose allopurinol), hydroxyl radical ablation was only effective when administered before the priming step (lipopolysaccharide) and was ineffective when administered later, prior to the activation step (heat shock). CONCLUSIONS: These findings suggest a novel role for the hydroxyl radical as a nonlethal intracellular signal in endothelial cell apoptosis. Moreover, the results support a role for programmed cell death in the pathogenesis of multiple organ dysfunction syndrome and suggest novel strategies for prophylaxis and therapy of the most common cause of death in surgical intensive care units.

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The homeostatic response of complex eukaryotes to the challenge of environmental stress includes the induction of several programs of gene expression; among them are those for the acute phase genes and those for the heat shock genes. In some systems, the heat shock response, which is often elicited by more severe stimuli, preempts the acute phase response, which is seen in response to less severe challenges, as well as constitutive gene expression. Nevertheless, each response appears to provide a natural selective advantage for survival of the organism in a toxic environment. However, when cultured porcine endothelial cells were exposed first to a nonlethal level of bacterial endotoxin lipopolysaccharide (LPS), an inducer of the acute phase response, and then, simultaneously to standard stimuli, which normally elicit a salutary heat shock response, the cells died manifesting a pattern of DNA fragmentation (nucleolysis) characteristic of programmed cell death (apoptosis). The treatment of LPS-exposed cells with cycloheximide to block protein synthesis reproduced the lethal apoptosis that had been elicited by the induction of heat shock gene expression. Therefore, the preemption of other programs of stress gene expression by the prioritized expression of heat shock genes is associated with apoptosis.

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