Cell biology of ischemia/reperfusion injury

Abstract

Disorders characterized by ischemia/reperfusion (I/R), such as myocardial infarction, stroke, and peripheral vascular disease, continue to be among the most frequent causes of debilitating disease and death. Tissue injury and/or death occur as a result of the initial ischemic insult, which is determined primarily by the magnitude and duration of the interruption in the blood supply, and then subsequent damage induced by reperfusion. During prolonged ischemia, ATP levels and intracellular pH decrease as a result of anaerobic metabolism and lactate accumulation. As a consequence, ATPase-dependent ion transport mechanisms become dysfunctional, contributing to increased intracellular and mitochondrial calcium levels (calcium overload), cell swelling and rupture, and cell death by necrotic, necroptotic, apoptotic, and autophagic mechanisms. Although oxygen levels are restored upon reperfusion, a surge in the generation of reactive oxygen species occurs and proinflammatory neutrophils infiltrate ischemic tissues to exacerbate ischemic injury. The pathologic events induced by I/R orchestrate the opening of the mitochondrial permeability transition pore, which appears to represent a common end-effector of the pathologic events initiated by I/R. The aim of this treatise is to provide a comprehensive review of the mechanisms underlying the development of I/R injury, from which it should be apparent that a combination of molecular and cellular approaches targeting multiple pathologic processes to limit the extent of I/R injury must be adopted to enhance resistance to cell death and increase regenerative capacity in order to effect long-lasting repair of ischemic tissues.

Figure 6.1

Figure 6.1 Major pathologic events contributing to ischemic (Upper Panel) and reperfusion (Middle Panel) components of tissue injury, with overall integrated responses to I/R injury summarized in the Bottom Panel. See text for further explanation. Modified from Sanada et al. (2011).

Figure 6.1

Figure 6.1 Major pathologic events contributing to ischemic (Upper Panel) and reperfusion (Middle Panel) components of tissue injury, with overall integrated responses to I/R injury summarized in the Bottom Panel. See text for further explanation. Modified from Sanada et al. (2011).

Figure 6.2

Total injury sustained by a tissue subjected to prolonged ischemia followed by reperfusion (I/R) is attributable to an ischemic component and a component that is due to reestablishing the blood supply. At the onset of prolonged ischemia two separate pathologic processes are initiated. The first are processes of tissue injury that are due to ischemia per se. The second are biochemical changes during ischemia that contribute to the surge in generation of reactive oxygen species and infiltration of proinflammatory neutrophils when molecular oxygen is reintroduced to the tissues during reperfusion particularly the initial phases. For a treatment to be effective when administered at the onset of reperfusion, reestablishing the blood supply must occur before damage attributable to ischemia per se represents a major component of total tissue injury. Therapeutic approaches that target pathologic events contributing to both the ischemic and reperfusion components of total tissue injury, such as ischemic or pharmacologic preconditioning, should be more effective than therapies administered when the blood supply is re-established, which limit only the progression of reperfusion injury. Modified from Bulkley (1987).

Figure 6.3

Tissue responses to ischemia/reperfusion are bimodal, depending on the duration of ischemia. Prolonged and severe ischemia induces cell damage that progresses to infarction, with reperfusion often paradoxically exacerbating tissue injury by invoking inflammatory responses. In the heart, shorter bouts of ischemia (5–20 minutes duration) induce myocardial stunning, wherein contractile function is initially impaired on reperfusion, but slowly improves, without progression to infarction and in the absence of significant inflammation. In sharp contras, prolonged exposure to subacute levels of ischemia without reperfusion may induce myocardial hibernation, wherein cardiac cells revert to a more ancestral metabolic phenotype in order to survive but with a cost of reduced mechanical function. In sharp contrast, short periods of ischemia (< 5 min) followed by reperfusion (ischemic conditioning) activate cell survival programs that limit the magnitude of injury induced by subsequent exposure to prolonged I/R.

Figure 6.4

Mechanisms of cell death in ischemia/reperfusion (I/R). I/R-induced necrosis generally occurs as a result of dysfunctional ion transport mechanisms, which causes cells to swell and eventually burst, effects that are exacerbated by plasma membrane damage. Release of proinflammatory mediators and damaged biomolecules initiates the influx of inflammatory cells such as neutrophils, which disrupt the extracellular matrix and cause damage to parenchymal cells by release of cytotoxic oxidants and hydrolytic enzymes. Apopotosis is a regulated form of cell death that causes cell shrinkage and condensation of the cytosol and nucleus, which eventually form apoptotic bodies. Because they are surrounded by cell membranes, apoptotic bodies can be engulfed and digested by phagocytes without evoking an inflammatory response. Autophagy provides a mechanism to remove damaged or senescent protein aggregates and organelles by enclosing them in membrane-lined vesicles called phagosomes which fuse with lysosomes containing enzymes that degrade the ingested material, usually without evoking an inflammatory response. While normally performing this “housekeeping” function, autophagy may also provide cells with a survival mechanism to withstand the deleterious effects of ischemia, by generating amino acids and fatty acids for cell function. However, when uncontrolled, autophagy contributes to ischemic cell death. While necrosis was once believed to occur from non-specific trauma or injury as a result of I/R, it now appears that postischemic infarction may also be attributable to programmed events that require a dedicated molecular circuitry that has been termed programmed necrosis or necroptosis. Necroptosis is initiated by TNF-like cytokines that activate RIP kinases to mediate necrosis via increased production of reactive oxygen species and calcium overload, which in turn modulate the mitochondrial permeability transition pore, leading to dissipation of the proton electrochemical gradient, with subsequent ATP depletion, further ROS production, and swelling and rupture of mitochondrial membranes.

Figure 6.5

Role of mitochondria in ischemia/reperfusion-induced cell injury. Opening of the MPT pore in the inner mitochondrial membrane is a critical event in the progression of cell death in response to I/R. Being inhibited by low pH, the MPT pore is kept quiescent during ischemia. However, upon reperfusion the huge increases in mitochondrial Ca²⁺, coupled with the ROS burst induce opening of the MPT pore. When this pore opens, H⁺ moves back into the matrix, thereby dissipating the Δψ_m, uncoupling the electron transport chain and inhibiting ATP synthesis. In addition, water enters the mitochondria through its osmotic gradient causing the mitochondria to swell and even rupture. There are several mitochondrial sources of ROS including the electron transport chain, p66Shc, mitochondrial K_ATP channels, and monoamine oxidases. Mitochondria are dynamic organelles that form tubular, intercommunicating networks that are linked to the cytoskeleton and undergo cycles of division (fission) and fusion. Alterations in mitochondrial morphology occur when these latter two processes become unbalanced, with loss of fission resulting in the appearance of large networks of fused mitochondria, while excessive fission leads to small, fragmented mitochondria. Because fission is initiated under conditions associated with I/R, such as low ATP levels and increase mitochondrial ROS production, and excessive mitochondrial fission is a required step for extrinsic apoptotic cell death, this process may contribute to the pathogenesis of postischemic cell death. Mitochondrial fission also contributes to fragmentation of these organelles in endothelial cells exposed to H/R and may thus contribute to endothelial dysfunction in postischemic tissues.

Figure 6.6

Functional roles and target genes for miRNAs implicated in ischemia/ reperfusion and preconditioning. See text for further explanation. Modified from Abdellatif (2012).