Reperfusion injury, the paradoxical tissue response that is manifested by blood flow-deprived and oxygen-starved organs following the restoration of blood flow and tissue oxygenation, has been a focus of basic and clinical research for over 4-decades. While a variety of molecular mechanisms have been proposed to explain this phenomenon, excess production of reactive oxygen species (ROS) continues to receive much attention as a critical factor in the genesis of reperfusion injury. As a consequence, considerable effort has been devoted to identifying the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Of the potential ROS sources described to date, xanthine oxidase, NADPH oxidase (Nox), mitochondria, and uncoupled nitric oxide synthase have gained a status as the most likely contributors to reperfusion-induced oxidative stress and represent priority targets for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. Although all four enzymatic sources are present in most tissues and are likely to play some role in reperfusion injury, priority and emphasis has been given to specific ROS sources that are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The possibility that multiple ROS sources contribute to reperfusion injury in most tissues is supported by evidence demonstrating that redox-signaling enables ROS produced by one enzymatic source (e.g., Nox) to activate and enhance ROS production by a second source (e.g., mitochondria). This review provides a synopsis of the evidence implicating ROS in reperfusion injury, the clinical implications of this phenomenon, and summarizes current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.

Reperfusion injury and reactive oxygen species: The evolution of a concept.

Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-κB. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier fun...

https://www.physiology.org/doi/pdf/10.1152/ajpcell.2001.280.4.C719

Oxidant stress and endothelial cell dysfunction.

Ischaemia-reperfusion injury is a complex phenomenon often encountered in surgical practice. The consequences of such injury are local and remote tissue destruction, and sometimes death. Several different processes have been implicated. This review discusses the pathology of such injury, the mechanisms of free radical production, and the role of neutrophils and endothelial factors in ischaemiareperfusion. Finally, several mechanisms that limit ischaemia-reperfusion injury are discussed and a number of novel Therapies presented

Ischaemia-reperfusion injury

Neutrophils respond to myocardial ischemia-reperfusion in a manner similar to the bacterial invasion of a host. The inflammatory-like response that follows the onset of reperfusion involves intense interactions with the coronary vascular endothelium, arterial wall, and cardiomyocytes in a very well-choreographed manner. Neutrophils have been implicated as primary and secondary mediators of lethal injury after reperfusion to coronary vascular endothelium and cardiomyocytes. The involvement of neutrophils in the pathogenesis of lethal myocardial injury has been inferred from (1) their presence and accumulation in reperfused myocardium in temporal agreement with injury induced, (2) the armamentarium of toxic agents such as oxidants and proteases that are released by neutrophils in reperfused myocardium, (3) responsivity to (recruitment by and/or activation by) inflammatory factors released by reperfused myocardium, and (4) inhibition of lethal post-ischemic myocyte or endothelial cell injury by strategies that interdict neutrophil interactions at any number of stages. However, whether neutrophils are directly involved in the pathogenesis of lethal reperfusion injury in the myocardium, are just pedestrian (first) responders to inflammatory signals released after the onset of reperfusion, or are important to an early but not clinically important phase of pathology are still points of controversy. As with the general area of myocardial protection itself, the failure to reproduce the salubrious effects of anti-neutrophil therapeutic strategies and to successfully translate these strategies into clinical practice has not only fueled the debate, but has jeopardized the further pursuit of myocardial protection therapeutics to improve post-ischemic outcomes. This review will describe the molecular responses of neutrophils to ischemia-reperfusion, discuss the cellular and tissue damage inflicted either directly or indirectly by these white cells, and discuss the physiological impact of interdiction of neutrophil-mediated interactions with myocardial cells at various levels on lethal post-ischemic injury. In addition, it will discuss the arguments for and against the involvement of neutrophils in responses to ischemia-reperfusion in experimental models, and the failure to translate experimentally successful therapy into clinical practice.

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Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury

The initial step in extravasation of neutrophils (polymorphonuclear leukocytes [PMNs]) to the extravascular space is adherence to the endothelium. We examined the effect of oxidants on this process by treating human endothelial cells with H2O2, t-butylhydroperoxide, or menadione. This resulted in a surface adhesive for PMN between 1 and 4 h after exposure. The oxidants needed to be present only for a brief period at the initiation of the assay. Adhesion was an endothelial cell-dependent process that did not require an active response from the PMN. The adhesive molecule was not platelet-activating factor, which mediates PMN adherence when endothelial cells are briefly exposed to higher concentrations of H2O2 (Lewis, M. S., R. E. Whatley, P. Cain, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman. 1988. J. Clin. Invest. 82:2045-2055), nor was it ELAM-1, an adhesive glycoprotein induced by cytokines. Oxidant-induced adhesion did not require protein synthesis, was inhibited by antioxidants, and, when peroxides were the oxidants, was inhibited by intracellular iron chelators. Granule membrane protein-140 (GMP-140) is a membrane-associated glycoprotein that can be translocated from its intracellular storage pool to the surface of endothelial cells where it acts as a ligand for PMN adhesion (Geng, J.-G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. 1990. Nature (Lond). 343:757-760). We found that endothelial cells exposed to oxidants expressed GMP-140 on their surface, and that an mAb against GMP-140 or solubilized GMP-140 completely blocked PMN adherence to oxidant-treated endothelial cells. Thus, exposure of endothelial cells to oxygen radicals induces the prolonged expression of GMP-140 on the cell surface, which results in enhanced PMN adherence.

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Oxygen radicals induce human endothelial cells to express GMP-140 and bind neutrophils.

Oxidant-induced damage to the intima of pulmonary and systemic vessels is thought to be an important mechanism of injury in a variety of syndromes of vascular damage. Hydrogen peroxide (H2O2) is an active oxygen metabolite that may induce intimal injury by cytolytic attack or by inducing biochemical and functional alterations in the endothelial cells (EC); however, mechanisms involved in noncytolytic perturbation of EC are largely unknown. We found that H2O2 stimulated the synthesis of platelet-activating factor (PAF) by primary cultures of bovine pulmonary artery endothelium (BPAEC) and by human umbilical vein endothelium (HUVEC). In each cell type the incorporation of [3H]acetate into [3H-acetyl]PAF was concentration- and time-dependent and was temporally dissociated from severe plasma membrane disruption and cytolytic cell injury; the newly synthesized PAF remained associated with the EC. H2O2 caused permeabilization of EC to 45Ca2+ and an increase in intracellular Ca2+, suggesting that a transmembrane Ca2+ flux is the signal that initiates PAF synthesis. H2O2 also induced the endothelial cell-dependent adhesion of neutrophils to HUVEC monolayers. This response was rapid, with an onset within minutes and a subsequent time course that paralleled the time course of PAF accumulation, and was dependent on extracellular Ca2+ but not on de novo protein synthesis. These studies demonstrate that H2O2 can induce two rapid activation responses of endothelium, PAF synthesis and EC-dependent neutrophil adhesion, events that may be important in physiologic and pathologic inflammation.

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P Cain

Papers

Hydrogen peroxide stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion.