reperfusion injury

International Journal of Cardiology 86 (2002) 41–59 www.elsevier.com / locate / ijcard

Oxidative stress and neutrophil activation—the two keystones of ischemia / reperfusion injury Karol A. Kaminski, Tomasz A. Bonda, Janusz Korecki*, Wlodzimierz J. Musial Department of Cardiology, Medical Academy of Bialystok, ul. M. Sklodowskiej-Curie 24 a, 15 -276 Bialystok, Poland Received in revised form 19 March 2002; accepted 6 April 2002

Abstract The widespread introduction of fibrinolytics and recently also PTCA in the treatment of myocardial infarction has changed the picture of modern cardiology. But this therapy also raises new problems and challenges. One of them is the occurrence of extensive tissue injury caused by reperfusion. Reinstitution of oxygen to the ischemic tissues initiates various processes leading to generation of reactive oxygen species (ROSs). Acting on the plasma membrane ROS damage its organization and release various proinflammatory agents. Different proteins, including receptors, ionic channels, transporters or components of transduction pathways are substrates of oxidation by ROSs. Their changed structure results in altered functioning and disruption of vital cellular processes. Another key factor of reperfusion injury is activation and infiltration of infarcted area by polymorphonuclear leukocytes (PMNs). Multiple studies identified consecutive stages of PMN activation and substances being involved in it. Main interest lies in cellular adhesion molecules, particularly selectins and b2 integrins, as their antagonists were repeatedly found to diminish neutrophil activation and infarct size. Nevertheless new publications strike at the foundations of the established order and confront the relation between neutrophil infiltration and infarct size. PMNs are linked by close ties to other cells involved in inflammatory response. Seemingly also in cardiac ischemia–reperfusion injury, the activity of neutrophils is modulated by lymphocytes and macrophages. The article describes mutual interactions between different factors involved in the reperfusion injury that may enable preparing new treatments, hopefully as effective and successful as reperfusion therapy.  2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Reperfusion injury; Oxidative stress; Reactive oxygen species; Neutrophils; Cytokines; Myocardial ischemia

1. Introduction Cardiovascular diseases are the most common cause of death in the western world. According to the WHO MONICA Project [1], they account for around 40% of all-cause mortality. Myocardial infarction plays a major role in this mortality, but changes in therapy over the last 20 years have markedly im-

*Corresponding author. Tel.: 148-85-746-8656; fax: 148-85-7468604. E-mail addresses: [email protected] (J. Korecki).

proved prognosis. Introduction of Intensive Coronary Units was the first milestone. Next, the 1980s brought about probably the biggest breakthrough—the implementation of reperfusion therapy. Although the underlying mechanism was suggested by Herrick almost 90 years ago [2] and the possibility of reperfusion therapy was discussed more than 40 years ago [3], it was only in the late 1980s that this method was put into practice [4,5] all around the world, with the combined use of intravenous streptokinase and aspirin. The 1990s witnessed gradual improvement in terms of better fibrinolytics, adjuncts to therapy [such as gp IIb / IIIa inhibitors, low-molecular-weight

0167-5273 / 02 / $ – see front matter  2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 02 )00189-4

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heparin (LMWH) or angiotensin converting enzyme inhibitors (ACEIs)], and also the increasing availability of PTCA in the acute phase of the myocardial infarction [6]. All of these therapeutic actions considerably decreased mortality. The quarterly report of The Third National Registry of Myocardial Infarction from the December 1999 presents the overall death rate in this group as 10.2% [7]. However, there are still in many cases where the therapy does not meet expectations. Sometimes, despite early and apparently successful reperfusion, prognosis is poor due to the development of heart failure. In these cases many cardiologists turn their attention to the idea of a reperfusion injury. The concept of augmentation of cardiac necrosis after a period of ischemia followed by reperfusion was formulated after experiments in the 1960s [8]. Such a phenomenon was also observed during operations with cardio-pulmonary bypass, when the systolic function deteriorated after surgery despite restoration of the blood flow. For years this problem was a source of controversy [9] as it was very difficult to demonstrate that the observed pathological changes were indeed the result of reperfusion, rather than being caused by ischemia and only terminated after reperfusion [10]. Relatively recently, new experimental methods [11] have enabled demonstration of cardiac injury caused by the restoration of blood flow. Studies that revealed the possibility of containing the infarct area using substances applied after reperfusion were particularly convincing [12]. Unfortunately the frequently used term ‘‘reperfusion injury’’ is vague, and it is often used to name all the possible adverse changes that arise following the restoration of oxygenated blood flow. It could be divided into two major phenomena: 1. Incomplete tissue reperfusion on a microvascular level, despite patency of an epicardial artery (also called no-reflow or low-reflow phenomenon). (a) With poor flow in epicardial artery when assessed by angiography. (b) With good flow. 2. Myocardial injury caused by reoxygenation and stimulation of inflammatory processes. In many cases these phenomena follow one

another, presenting as early and late phases of the injury. During the former, the endothelium is the primary target and its dysfunction augments ischemic injury by activating platelets and leukocytes. Therefore it is frequently described as microvascular injury or dysfunction. During the latter, the proinflammatory cytokines in concert with migrating activated leukocytes cause direct damage to the myocardium. Despite different manifestations the underlying mechanisms are similar. In both, oxidative stress and leukocytes play the key roles.

2. Oxidative stress The hypothesis of free radical mediated injury of the myocardium during reperfusion is based on the observations, that: 1. Reperfusion induces impairment in myocardial function [13]. 2. Restoration of blood flow to the ischemic area results in excessive production of reactive oxygen species (ROS) [14,15]. 3. Antioxidant interventions diminish myocardial injury [16]. 4. Experimental application of oxidative equivalents induces similar disturbances as seen in vivo during reperfusion [17]. Electron paramagnetic resonance spectroscopy, the most widely approved laboratory method for directly measuring free radical formation, indicates that they are produced in the greatest amount during the first minute of reperfusion, although their overproduction lasts for tens of minutes [14,15]. The early phase of blood flow restoration seems to play a crucial role in future events. ROSs are mainly free radicals, which means that they have at least one unpaired electron, such as superoxide anion (O 2 2 ) or hydroxyl radical ? (OH ). Hydrogen peroxide (H 2 O 2 ) is not a radical, but plays an important role in oxidative processes. One-electron reduction of oxygen results in superoxide formation. This undergoes the catalytic process of dismutation, or spontaneously reacts with protons to produce H 2 O 2 . Once created superoxide may initiate a cycle of iron oxidation / reduction in which hydrogen peroxide is produced [18]. Oxygen-derived oxidative species are not the only

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substances taking part in reperfusion injury. Many other substances take part in the oxidative processes that are capable of injuring cardiac tissue. Nitric oxide (NO) derivatives are one of the most intensively examined in this context. Nitric oxide is synthesized in the endothelium by endothelial nitric oxide synthase (eNOS), in neutrophils by constitutive nitric oxide synthase (cNOS), and—especially during stress—by inducible nitric oxide synthase (iNOS). NO release is upregulated just after oxygenation of the ischemic myocardium [19,20]. Experiments on eNOS knockout animals shown that this enzyme is activated early and plays a protective role during ischemia / reperfusion [21]. Unlike eNOS contribution of iNOS is more complicated. It is possible that amounts of nitric oxide and peroxynitrite formed during late reperfusion are much higher than early after blood flow restoration because of upregulation of iNOS, which requires over 4–6 h and is maximal after 48 h [22,23]. This delayed augmentation of nitric oxide level increases tissue injury. As it follows from experiments of Wildhirt et al. iNOS selective inhibition has cardioprotective effects with respect to myocardial performance as well as coronary blood flow, cellular infiltration, extent of necrosis and infarct size [23]. Supportive for iNOS induced injury is work of Wang et al. [24]. In contrast Kanno et al. describe beneficial influence of iNOS superinduction on ischemia / reperfusion injury [25]. In a very recent article, Zingarelli et al. describe an experiment with iNOS knockout (iNOS KO) mice subjected to 1-h ischemia followed by 1-h reperfusion. iNOS KO mice, comparing to wild type, had more extensive myocardial injury, marked apoptosis and neutrophil infiltration, decreased expression of cytoprotective agents like heat shock protein 70 and IL-10, increased TNFa and IL-6. There also were differences in nuclear transcription factors AP-1 and NF-kB downregulation. The authors conclude that iNOS plays a putative role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction [26]. On the other hand Jones et al. did not reveal any influence of iNOS deficiency on the extent of necrosis and neutropil infiltration [27]. Similar results were also published by others [28]. The mechanisms underlying the contribution of NO to ischaemia / reperfusion injury involve many

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possible mechanisms. Its effects on leukocyte activation and platelet adherence will be absent in any investigation using the in vitro crystalloid-perfused animal heart. However, the effects on endothelium, including vasomotor tone and free radical generation persist. Detailed information regarding interactions of NO, endothelium and leukocytes is presented further in this review. NO scavenges superoxide, but on the other hand it forms peroxynitrite (ONOO 2), a potentially cytotoxic derivative. Peroxynitrite is synthesized mostly during first 2 min of reperfusion [19,29]. Experimental addition of a superoxide scavenger or NOS inhibitor results in higher recovery of contractile function, indicating that ONOO 2 exerts a depressive effect on cardiac performance [30]. Recent observations with cardioplegic solutions revealed that peroxynitrite may act as a cardioprotective and antineutrophil compound when administered in blood cardioplegia, although the opposite results were noted when crystalloid solution was used [31]. Despite being shown to exert deleterious effects, ONOO 2 in rather low, physiologically relevant concentrations, as mostly observed, is reported to be protective [32]. It was recently observed that it attenuates neutrophil adhesion to the endothelium and decreases expression of selectin-P [33]. During reperfusion the peroxynitrite inhibits xanthine oxidase activity and diminishes O 2 2 synthesis [34]. Sawicki and co-workers showed that ONOO 2 rapidly activates metalloproteinase-2, which is thought, among other actions, to impair mechanical function of cardiac muscle because of contractile apparatus proteins degradation [35,36]. Several of the cell types in cardiac tissue contain potential sources of reactive oxygen species. These cells are the cardiomyocytes, endothelial cells and neutrophils, as well as interstitial cells. Activated neutrophils are reported to be the main source of superoxide radicals during reperfusion [37]. After stimulatation by proinflammatory mediators, they are recruited to the site of injury and produce superoxide anions via the NADPH oxidase pathway. Inhibition of neutrophils adhesion by administration of anti-CD18 monoclonal antibodies or blocking of NADPH oxidase, attenuates superoxide release. Dismutation of superoxide, catalysed by the myeloperoxidase released from azurophilic granules, results in synthesis of hydrogen peroxide, hydroxyl radicals, and hypochlorous acid. In addition to this, the

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degranulation of neutrophils gives rise to the synthesis of other oxidants, as many enzymes involved in their formation are released. The main source of ROS in the cardiomyocyte is mitochondrial electron transporting system. The exact mechanism is not clear, but experimental inhibition of its activity at the level of NADH dehydrogenase (i.e., at the early step of the mitochondrial respiratory chain) significantly decreases the release of oxidative species during reperfusion. NADH-dehydrogenase and semiquinones are thought to be involved in O 2 2 synthesis [38,39]. Endothelial cells produce radical species in several ways. First, xanthine oxidoreductase, with dominant activity of dehydrogenase in physiological conditions, is converted into oxidase during ischemia, with subsequent superoxide production. In addition to the purine metabolizing path, the nitric synthase was reported to release ROS under stress conditions and endothelial cells as well as fibroblasts express xanthine oxidase activity during oxygen depletion and reintroduction.

2.1. Targets for oxidative injury ROS exert many deleterious effects on the myocardial tissue. They react with proteins, membrane lipids, and nucleic acids. Proteins are attacked primarily by hydroxyl radical. The most susceptible to oxidation are sulfhydryl groups. Modification of these may produce disulphide bond formation and intermolecular cross-linking or sulphoxide formation. Oxidation of proteins changes their structure and function. Oxidation of the backbone of the protein may even result in bond cleavage and molecule destruction. Many proteins playing a key role in homeostasis of the cardiomyocyte are modified during massive ROS release. Membrane ionic channels change their permeability to potassium, calcium and to the lesser extent to sodium. Na / Ca exchange was reported to be stimulated in the presence of ROS. Activity of ion pumps like Na,K-ATPase, CaATPase, or SR CaATPase was decreased after exposure to the oxidative stress [40]. Oxidative modification of contractile proteins may result directly in hemodynamic failure [41,42]. Proteins modified by free radicals are resistant to proteolytic degradation. Proteasome, a major intracellular proteolytic system

degrading oxidized forms of proteins, is reported to be inhibited due to oxidation during ischemia / reperfusion, thus increasing the cytosolic levels of modified proteins [43]. Destruction of nucleic acid by ROS has been reported in the setting of ischemia / reperfusion, especially mitochondrial DNA (mtDNA), as it is present in the direct proximity to the site of ROS formation. It has been attributed to ischemia / long-reperfusioninduced decreased activity of complexes I, III and IV of the respiratory chain, that are encoded by mtDNA. Lipids are another very important cell element that is damaged by oxidation. Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts [44,45]. A lipid oxidation reaction begins with the formation of a radical species from a non-radical fatty acid precursor. Hydrogen peroxide as well as perhydroxyl radical (HOO ? ) can extract bis-allylic hydrogen atom of an unsaturated fatty acid to form a lipid alkyl radical. Further oxidation by O 2 2 produces a peroxyl radical, which in the next step reacts with an unsaturated fatty acid. Prapagation of this radical chain reaction is essential for successive oxidative damage to lipids and membrane-attached proteins. Hydroperoxides and conjugated dienes are often measured as sensitive indicators of oxidative stress.

2.2. Antioxidative defense Under physiological conditions small amounts of ROS, produced as a consequence of electron transfer reactions in mitochondria, peroxisomes and cytosol, are scavenged by cellular defending systems. During ischemia / reperfusion the activity of these systems becomes reduced or even abolished. Antioxidants act by scavenging oxidative species and their precursors, inhibiting their formation and enhancing endogenous antioxidant defense. The main endogenous antioxidant is superoxide dismutase (SOD). Two forms are found within the cell (cytosolic ZnCu-SOD and mitochondrial MnSOD), and one form in the extracellular space, which further divides into subclasses depending on the affinity to heparan. The function of all SODs remains the same—they catalyse O 2 reduction to H 2 O 2 . 2 Transgenic animal experiments revealed a beneficial

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effect of overexpression of MnSOD on infarct size, but other investigations did not reveal any advantages. It has been noted numerous times that administration of SOD and catalase protects reperfused hearts from injury, when they are applied together. The primary target of their protective action seems to be calcium-balancing mechanisms. Individual administration of SOD or catalase does not always show a satisfying amelioration. Despite the inconsistency of the results of previous investigations dealing with SOD and catalase, an attempt has been made to use this approach to treat or prevent reperfusion injury. Based on previous observations of better outcome after infarction in rats overexpressing SOD, transfection of animals with a gene for extracellular SOD resulted in powerful protection against ischemic injury [46]. The activity of Mn-SOD declines under the influence of peroxynitrite. Superoxide reacts faster with NO than with SOD, thus reperfusion induces rapid increase in ONOO 2 concentration. Further, nitration of one tyrosine residue of the enzyme by ONOO 2 results in inactivation of SOD [47]. The process of nitration is promoted by manganese [48]. Catalase, a membrane bound peroxisome enzyme, although not appearing to play a crucial role in the salvage of myocardium from reperfusion injury, has been reported to exert its positive role. Catalase has a lower H 2 O 2 quenching activity than the next antioxidant enzyme discussed, glutathione peroxidase. Glutathione peroxidase catalyzes the peroxidation of H 2 O 2 in the presence of reduced glutathione (GSH) to form H 2 O and oxidized glutathione (GSSG). The GSSG recycles back to give GSH by glutathione reductase, which requires NADPH, mainly recruited from the hexose monophosphate shunt. In the setting of ischemia concentration of NADPH dramatically declines and restoration of blood supply does not recover preischemic NADPH levels. Also, thiol groups that take a superior position in balancing whole-cellular redox potential (closely interconnected to GSH / GSSG turnover) are deprived of their reducing properties. Thus, low NADPH and high GSSG concentrations attenuate glutathione peroxidase antioxidant activity during reperfusion [49]. Two more antioxidants are of interest, because they may be easily enhanced in the living organism.

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These are vitamins C and E. Vitamin E is a lipidsoluble molecule, so it effectively prevents lipid oxidation. It has been shown that vitamin E-treated rats had better preserved contractile function and decreased infarct size. In humans there is no that clear evidence of its positive influence. One randomized investigation—CHAOS—demonstrated a marked reduction in non-fatal myocardial infarction in subjects treated with vitamin E as compared with the placebo group [50], however other clinical trials did not reveal any significant influence of this type of treatment [51–55]. Recent observations on rabbits have revealed a significant protective effect of preventive food supplementation with vitamin C. Animals receiving ascorbic acid were less susceptible to tissue injury during ischemia / reperfusion. But the beneficial role of vitamin C has recently been put to a hard test. Opposing evidence was presented indicating better recovery of contractile function in mutant, vitamin C-deficient rats compared with normal animals. What is also worthy of attention is that similar levels of free radical release were observed during reperfusion [56]. This experimental data was supported by the clinical observation that usage of vitamin C prior to PTCA in acute myocardial infarction had no lowering effect on urine level of 8-epi prostaglandin F2a (an indicator of oxidative stress in vivo during myocardial reperfusion) [57]. It has been speculated that lack of antioxidative effect of vitamin C administration may result from the fact that subjects entitled to the study already had a maximal antioxidative level of vitamin C even without additional treatment [58]. Thus further investigations are required to evaluate these conflicting results. Some drugs widely used in clinical practice possess antioxidant properties. For example the beta adrenoreceptor blocker metoprolol is said to diminish lipid peroxidation and enhance reduced glutathione concentration [59] and another—carvedilol acts as free radical scavenger and diminishes oxidative stress [60]. Another often used drug amiodarone is reported to possess antioxidative properties, though its significance has not been established yet [61]. The targeted use of antioxidants is not often in clinical practice as for now. It may be related to fact, that despite of many attempts there is no drug with proven and evident favorable influence on outcome. Among

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many others probucol decreases oxidative stress and reduces restenosis after coronary balloon angioplasty [62] and exerts beneficial effect on survival after myocardial infarction in rats, probably in the mechanism of cardiac fibrosis reduction, diminishing oxidative stress and expression of pro-inflammatory cytokines, however further investigations are required [63]. N-Acetylcysteine has been administered to 20 patients with acute myocardial infarction and shown a slight plausible effect, and yet it was not further clinically tested on larger groups [64]. Another aspect of reperfusion tissue injury is related to programmed cell death. Oxidative stress and metabolic imbalance during ischemia and reperfusion evoke cytoplasmic calcium overload. This is mainly due to increased release of calcium from the sarcoplasmic reticulum by ryanodine channels and depressed function of Ca-ATPase. As mitochondria are the main organelles capable of balancing calcium homeostasis in these conditions, calcium is stored in the mitochondrial matrix. If excessive and lasting long enough, oxidation of inner membrane proteins and lipids, plus calcium overload and other metabolic alterations are reported to evoke mitochondrial permeability transition (MPT) believed to play a key role in programmed cell death [65,66]. Leakage of cytochrome c is an important activator of the caspase pathway leading to apoptosis. Costunolide induces apoptosis by ROS-mediated mitochondrial permeability transition and cytochrome c release [67]. Interestingly, H 2 O 2 and O 22 trigger distinct apoptotic signaling pathways in cardiomyocytes. There is another pathway to apoptosis that is related neither to cytochyome c release nor to MPT. It is inhibited by Bcl-2, but promoted by membrane potential alterations. This pathway is p53 dependent, and is also proposed to be mediated by ROS. It has been suggested that Bcl-2 functions as an antioxidant to prevent apoptosis. It may decrease lipid peroxidation and increase the cell resistance to ROS [68]. Bcl-2 may regulate the opening of MPT pores and inhibit mitochondrial dependent apoptosis [69,70]. However, an opposing proposal indicates that Bcl-2 can protect cells from death independently of ROS, because threatened cells can be rescued by Bcl-2 effectively, even in the absence of ROS [71]. Metabolic pathways related to Bcl-2, including regulation of caspase-3

activity, is a potential new subject for developing new antioxidtive and antiapoptotic therapy strategies and first attempts in this direction have already been made [72]. Transcription factors, AP-1 and NF-kB, are activated by ROS in response to cardiac ischemia / reperfusion [73]. These may promote several pathways connecting apoptosis and inflammatory responses during ischemia / reperfusion, although the precise biological response to the activation of these factors is under intensive investigation. Among the possible effects of NF-kB activation by hypoxia is upregulation of TNFa and interleukin-6 discussed further in this review [74,75]. NF-kB in the settings of hypoxic environment requires ROS for its activation and antioxidants such as pyrrolidine dithiocarbamic acid or N-acetylcysteine prevents NF-kB activation, as well as TNFa expression. Similar action was observed when rotenone, an inhibitor of mitochondrial complex I, was administered [74]. These may be the possible target for future therapeutic interventions. As oxidative mediators take part in many different signaling and metabolic pathways it is very difficult to understand these mechanisms. Recent years have brought new experimental tools like genetically modified animals, which opened a new door enabling us to deepen our knowledge in this field.

3. Neutrophil activation Infiltration of the infarcted tissue by polymorphonuclear leukocytes (PMNs) was already described in late 19th century [76]. Long before the reperfusion era inflammation was considered to be a pivotal point in myocardial infarction. Successful attempts to contain the damage in experimental setting raised new hopes [77]. But the first clinical trial brought disastrous results. Application of methylprednisolone caused infarct size extension, increased incidence of ventricular arrhythmias, hence increasing mortality [78]. Later, other studies have proved that neutrophil depletion reduces both ischemic [79] and reperfusion injury [80]. Papers that followed and have described time-dependent migration of leukocytes in detail [81], indicated that during first hours of reperfusion the intravascular compartment contains the greater part of

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the activated leukocytes, whereas migration and hence direct neutrophil–myocyte contact, occurs predominantly later—after 4–6 h. These results, however, are in opposition to others, stressing the importance of the early phase, and showing that after first hours the damage has already been done. So far, both the course of late reperfusion injury and contribution of neutrophils are not clear.

4. Early reperfusion injury Within moments of re-establishing oxygenated blood flow, emerging oxygen radicals directly increase the adherence of the neutrophils [82]. At the same time they act on arachidonic acid in cell membranes and LDL particles to produce F2-isoprostanes [83] which modulate the function of platelets [84], act as potent vasoconstrictors [85], and trigger very rapid adhesion of neutrophils to fibrinogen [86]. During first moments the most important events happen between neutrophils and endothelium

4.1. Endothelium–neutrophil interaction Functional studies on endothelium have demonstrated the particular vulnerability of endothelial cells to hypoxia. Moreover, subsequent reperfusion brings about further damage—within 150 s of re-opening the artery, the release of nitric oxide (NO) from the endothelium has markedly decreased [87]. Nitric oxide inhibits platelet aggregation and degranulation by direct action on platelet GMP-cyclase, and decreases expression of adhesion molecules on activated endothelium [88,89], leukocytes [90] and myocytes [91], hence attenuating neutrophil mediated injury to endothelium and myocytes. It inhibits also oxygen radical generation by neutrophils by direct action on membrane-bound NADPH oxidase [92], and attenuates neutrophil-mediated myocardial contractile dysfunction [93]. Deficiency of such a strong inhibiting agent promotes neutrophil activation and increases tissue injury [90–92]. The dysfunctional endothelium fails to attenuate leukocyte activation by the release of nitric oxide and adenosine, and fails to scavenge free radicals or produce EDHF. On the contrary, hypoxia and reoxygenation induce degranulation of Weibel–Palade bodies and expression of

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selectin-P (CD62 P) and platelet activating factor (PAF) on the luminal surface [94] of the endothelium, which augments neutrophil adhesion and again enhances microvascular injury [95]. These finding have been substantially supported by studies using eNOS knockout mice [96] and liposome-mediated eNOS transfection [97]. Directly after reperfusion profound changes also in the shape of the endothelial cell because of edema and a drop in intracellular pH occur, what in turn promotes contraction of the cytoskeleton in the endothelial cells, significantly narrowing the lumen of the vessels [98]. Hence activated neutrophils adhere together with activated platelets to the damaged vessel wall, and due to their rigidity as well as diminished capillary diameter, they clog the microvasculature [99] and considerably reduce tissue perfusion, creating a pathophysiological basis for noreflow phenomenon [100]. Platelet activation together with endothelial damage contributes to leukocyte recruitment and migration both by secretion of chemoattractants like PAF, PF4 or IL-8 [101] and increasing adherence [102]. Neutrophil–endothelium interaction can be suppressed by ischemic preconditioning. This acts both by reduction of expression of CD11b on granulocytes [103] and by protecting the functions of the endothelium, by inducing endothelial nitric oxide synthase and NFkB [104]. Following stages of neutrophils activation were described in detail elsewhere [105–107] and are summarized in Fig. 1.

4.2. Neutrophil recruitment After first reports about beneficial effects of neutrophil depletion during experimental infarction and reperfusion in laboratory animals [80,108], clinical trials followed, showing that leukocyte depletion may be beneficial during heart surgery [109,110]. The papers exploring the early stages of neutrophil recruitment demonstrated the key position of the selectin-P (Psel) and hinted at therapeutic potential of inhibiting leukocyte rolling thus attenuating neutrophil recruitment and myocardial injury within few hours after reperfusion [111,112]. Further studies, however, showed no difference in myocardial injury occurring during first 24 h in selectin-P devoid mice,

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Fig. 1. Stages of neutrophil activation. Chemotactic agents mobilize leukocytes in the circulation and prepare them for interaction with the endothelium. Substances acting on the endothelial cells increase expression of selectins, promote rolling, the vital stage of neutrophil activation. Granules containing CD11b / CD18 are then incorporated in the PMN’s membrane, intercellular adhesion molecules (ICAMs) are upregulated on the endothelium, and firm adhesion may occur. Diapedesis follows due to expression of platelet–endothelial cell adhesion molecule (PECAM-1). Direct myocyte–neutrophil contact, mediated by CD11b / CD18 and ICAM-1, leads to myocardial injury. Degranulation occurs at any place when integrins CD11b / CD18 are stimulated.

despite marked reduction of leukocyte accumulation [113,114].

4.3. Neutrophils as a source of bioactive substances Activated PMNs are one of sources of two major proinflammatory cytokines: IL1b and TNFa. These potent chemoattractants [115] originate, however, predominantly from residing macrophages [116] and mast cells [117], but also by lymphocytes, fibroblasts, smooth muscle, and even cardiomyocytes [118]. TNFa induces PMN adhesion and degranulation,

stimulates NADPH oxidase, and enhances expression of IL-2 receptors and expression of ICAM-1 on endothelium [119]. It stimulates expression of IL-1, IL-2, IL-6 and of PAF receptor [117]. Other features crucial for the late phase of reperfusion injury are the induction of myocyte apoptosis [118] and the depression of the contractile function [120]. Secretion of chemotactic agents occurs early during ischemia. It mobilizes PMNs in the entire circulation causing leukocytosis. Patients with myocardial infarction who present with higher white blood cells count are prone to ischemic episodes, and have generally poorer prognosis [121].

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Degranulation of neutrophils, apart from enhancing oxidative stress, results in increased tissue activity of elastase and collagenase, as well as augmented formation of active eicosanoids. Phospholipase A2 releases arachidonic acid from the plasma membrane. Cyclooxygenase generates thromboxanes A2 and B2, with pronounced vasoconstricive, platelet activating and even pro-apoptotic effects [122]. 5-Lipoxygenase in PMNs is predominant source of leukotrienes, powerful chemoattractants [123],which proinflammatory effects have been revealed clearly in pathology of asthma and allergy. Their production, both during acute coronary syndromes and heart surgery, is significantly increased [124,125]. Vasoconstriction, increased permeability and tissue edema, as well as chemotaxis of different types of granulocytes are major features of these substances [126]. LTB4, the most prevalent as well as the most stable form, also has other clinically important effects. These can be observed during ischemia and reperfusion, for example direct negative inotropic effect [127] or induction of ventricular arrhythmias [128], which are frequently being considered as one of the clinical features of reperfusion. However, whilst some studies have shown beneficial consequences of lipoxygenase– cycloxygense inhibition [129], others with LT receptor inhibitors have failed to reveal a cardioprotective effect [130]. PAF released by activated neutrophils may also induce common post-reperfusion arrhythmias [131]. Its antagonists have been found to have antiarrhythmic effect in animal models [132]. It is also an important factor also in regulation of LDL oxidation, where its increased activity attenuates oxidation [133]. Exogenous PAF acetylhydrolase (an enzyme which cleaves it to lyso-PAF) diminishes infarct area, attenuates contractile dysfunction and inhibits neutrophil–endothelium interactions hence limiting injury [134]. Proteases secreted during neutrophil degranulation, particularly elastase and collagenase, damage both the vessel wall and myocardium. It is elastase that predominantly contributes to the injury, due to its highly cationic nature which alters membrane charge distribution [106]. Recent studies have found a correlation between the degree of activation of, and tissue infiltration by, neutrophils (measured by myeloperoxidase activity)

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and the extent of apoptosis in reperfused tissue [135,136]. However this effect should be investigated further to determine precisely which pathways and substances are involved in apoptosis brought about by PMNs.

4.4. Lymphocytes Involvement of lymphocytes in the early phase of the reperfusion injury until recently has not been considered at all. The requirement for contact with antigen bound to major histocompatibility complex (MHC) protein, to activate T-helper cells, seemed to exclude possibility of early activation of lymphocytes. However, an alternative pathways of lymphocyte activation has been described [137]. Nonetheless, papers on the contribution of lymphocytes to myocardial ischemia / reperfusion injury are scarce, though interesting work has been done on renal and liver models. Clavien et al. showed activation of lymphocytes by oxygen radicals [138] during reperfusion of rat liver. In a similar experimental setting in the mouse, Zwacka et al. demonstrated the induction of inflammatory responses, including neutrophil accumulation, by CD4(1) lymphocytes [210]. Another link between activation of lymphocytes and early neutrophil mediated injury is provided in the paper by Rabb et al. Investigating a renal ischemia–reperfusion model, they demonstrated distinctly better renal function and smaller neutrophil infiltration in CD4 / CD8 knockout mice [139]. Kokura et al. showed that hydrogen peroxide produced by postanoxic endothelial cells stimulates T cells, which in turn enhance neutrophil adhesion to endothelium [140]. Therefore lymphocytes are very likely to be another modulator of neutrophil activation after reperfusion. However it is difficult directly to transfer directly these findings to myocardial ischemia / reperfusion, and there is clearly a need for further study in this field. A hypothesis of involvement of lymphocytes in pathogenesis of acute coronary syndromes, has been strongly supported by a study showing that CD4(1) T-cells from unstable angina patients may directly cause lysis of endothelial cells (HUVEC) [141]. Whether such activation plays important role in endothelial injury during reperfusion, so far one may only speculate. Studies of immunosuppressive agents, like cyclo-

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sporin A (CsA) and FK-506, provide new very interesting information. When administered after the onset of ischemia, decreased myocardial injury after reperfusion [12]. Moreover, the calcineurin, a calcium-dependent protein phosphatase inhibited by both agents, is involved not only in cardiac hypertrophic signaling [142], but also in the myocyte apoptosis induced by beta-adrenergic stimulation [143]. Another study [144] provided evidence for suppression by CsA of neutrophil activation and migration during reperfusion. This resulted in diminished myocardial necrosis, increased myocardial contractility, reduced serum TNFa levels and decreased ICAM-1 expression in myocardium. However it is not clear whether this was the direct effect of cyclosporin A on granulocytes, cardiomyocytes, or if it was mediated by lymphocytes.

4.5. Macrophages Involvement of macrophages in the progress of atherosclerosis is well known. Their impact during the healing phase of myocardial infarction is also well documented [145]. Whether they play a role in the early phase of reperfusion injury, and exert a negative effect, is despite good theoretical basis, unexplored. Macrophages resident in cardiac tissue are considered to be major source of IL-1b [146] and TNFa [116] early during reperfusion. Interleukin 1b, acting as the ‘‘first line’’ cytokine, and known to cause fever and increase prostaglandin formation, stimulates chemotaxis of neutrophils [147] but may also induce apoptosis by acting in concert with oxygen radicals [148]. Macrophages along with endothelium, smooth muscle cells and lymphocytes are the source of interleukin-6 [149], which induction of interleukin-6 during reperfusion injury has been well documented by both experimental [150] and clinical reports [151]. This prototypic pleiotropic cytokine, with both inflammatory and anti-inflammatory properties [152], primes the oxidative burst in neutrophils and monocytes [153], and induces ICAM-1 expression on myocytes [154], facilitating direct neutrophil– myocyte contact and promoting late reperfusion injury. On the other hand, it may exert cytoprotective effects via stimulation of gp130 receptor [155]. Also, recruitment of monocytes in the ischemic and reperfused myocardium is well documented [156]

and a recent study that found predominant localization of TNFa in newly recruited macrophages, presented the opinion that they are an important part both of the neutrophil activation cascade and ischemia reperfusion injury [157]. Another study has provided a link between the two mononuclear leukocytes: migrating monocytes secrete TNFa, which in turn promotes migration and extravasation of activated lymphocytes [158].

5. Late reperfusion injury Late reperfusion injury in humans is thought to follow reperfusion by 4 to 6 h, and is usually related to late myocyte death [106]. However, changes characteristic for no-reflow phenomenon may not only persist into the late phase, but may even aggravate [159]. Furthermore, direct myocyte injury causes tissue edema and compresses the microvasculature, further decreasing myocardial perfusion. Zhao et al. underlined the importance of late reperfusion injury, where observed early recovery of contractile function dissipated during further observation, and progressive extension of myocardial infarction was demonstrated between 6 and 24 h. Peak neutrophil accumulation was observed after 24 h, contrasting with the previous studies of Dreyer et al. [160]. This observation delivers a possible explanation why single doses of adhesion molecule antagonists applied before reperfusion, despite limiting myocardial injury during the early phase, fail to do so in the late phase (72 h) [161]. Nonetheless the importance of the late reperfusion injury is questioned by the papers showing that predominant part of the injury takes place during first hour. Integrins aLb2, also known as CD11b / 18 or membrane adhesion complex (MAC), are the main counterpart receptor for intercellular adhesion molecule-1 (ICAM-1). The latter is induced in heart after ischemia and reperfusion. Its expression, not only promotes migration, stimulates production of superoxide, IL8 and TNFa [162] but also gives basis to direct neutrophil–myocyte interaction and cytotoxicity [154]. Many studies have described the cardioprotective effects of inhibition of ICAM-1 [163,164] or its ligand CD18 [165]. Similar outcomes were demonstrated in ICAM-1 and CD18 deficient mice [166].

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These results, however, have been challenged by studies that used prolonged reperfusion resembling that in humans after primary PTCA or thrombolysis. Although antibodies against CD18 still provided protection [167], neither inhibition of ICAM [161], nor ICAM-1 [168] deficiency, limited infarct size after long-lasting reperfusion. In the latter study, Metzler et al. found sustained inhibition of neutrophil infiltration in ICAM-1 deficient animals, but reduction of the infarct size was observed only at the early phase of reperfusion, whereas after 1 or 3 weeks the scar size was similar in both types of animal. These results are supported by a study by Briaud et al., where mice deficient in both P-selectin and ICAM-1 had a profound decrease in neutrophil migration, but the infarct size did not differ between the two groups after 3 or 24 h of reperfusion [169]. Correspondingly Jones et al. documented that in genetically modified diabetic mice, despite reducing infiltration of neutrophils, P-selectin blockade failed to limit infarct size at 2 h of reperfusion [170]. These facts imply either that despite inhibition of common pathways of neutrophil activation, additional mechanisms emerge which lead to myocyte injury, or other factors play the primary role in the late reperfusion injury. Surprisingly, even deficiency of NADPH oxidase, the enzyme which is allegedly mainly responsible for neutrophil-mediated oxidative stress [37], does not protect the myocardium from the ischemia–reperfusion injury [171]. A study that demonstrated inhibition of neutrophil accumulation after myocardial ischemia / reperfusion by trimetazidine [172] raised another issue. Williams et al. showed that despite inhibition of myocardial infiltration, trimetazidine did not have any effect on intradermal accumulation of neutrophils in response to exogenous LTB4 or IL-8. It is plausible that myocardial neutrophil infiltration in certain experimental conditions is merely a marker of the injury and not necessarily the cause.

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expression of ICAM-1 on myocytes and markedly ameliorating neutrophil infiltration [174]. It strongly affects monocytes, inhibiting their production of IL1a, IL-1b, TNFa, IL-6 and IL-8 after LPS-stimulation [175]. Increased expression of IL-10 is observed in canine models after 5 h, and the peak follows at 96–120 h after reperfusion [173]. TGFb1 is another substance with a predominantly cardioprotective effect. Despite being potent chemoattractant [176], the overall effect of TGFb1 on reperfused myocardium is clearly protective [177]. TNFa plays special role in this balance between pro- and anti-inflammatory agents. On one hand it is one of the most potent substances increasing the chemotaxis of neutrophils [115], transcription of cytokines [117], and expression of adhesion molecules [119]. Acting on its receptors, it brings about apoptosis. The depression of contractile function, also produced by TNFa [178], is a part of clinical picture of sepsis [179], congestive heart failure [187,180] and particularly cardiomyopathy [181]. Its expression is up-regulated after ischemia and reperfusion [182,183]. Persistently elevated levels of TNF after myocardial infarction identify patients at increased risk of a recurrent coronary event [184]. On the other hand new, protective effects of TNF have been described. TNF-deficient mice were shown to develop much larger infarcts than the corresponding wild types [185]. There are several explanations for this phenomenon [186]. By activating transcription NF-kB-dependent proteins, TNFa increases expression of the cytoprotective genes involved in cellular growth, proliferation and survival [187,188]. Moreover pre-treatment with TNF protects from reperfusion injury by induction of manganese superoxide dysmutase [189]. The many functions of TNFa require further study before they can be successfully targeted by therapeutic interventions. Fig. 2 presents the most important interactions that occur during ischemia and reperfusion.

5.1. Cytokine balance In the late phase of reperfusion, the anti-inflammatory cytokines are secreted, creating a counter-balance for the early response, suppressing leukocyte activation. Interleukin-10, in this case secreted mainly by CD51 T lymphocytes [173], plays a fundamental role. This cytokine exerts a strong cardioprotective effect by inhibiting synthesis of TNFa attenuating

6. Possible therapeutic interventions limiting leukocyte activation Adenosine is one of very few agents in which the capability to limit reperfusion injury has been confirmed by in vivo trials on humans. In the experimental setting it reduces injury of the vascular endo-

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K. A. Kaminski et al. / International Journal of Cardiology 86 (2002) 41–59

Fig. 2. Schematic diagram of activation and inhibition pathways in reperfusion injury.

thelium, accumulation of PMNs and infarct size [190]. Although the AMISTAD trial did not find any changes in mortality and morbidity, it was documented that peripherally infused adenosine during thrombolysis results in a reduction of infarct size [191]. Another trial showed that intracoronary administration of 4 mg of adenosine just before reperfusion during the primary PTCA prevents no-reflow, attenuates LV dysfunction and improves the clinical course of the disease [192]. Nevertheless, further careful evaluation of dosage and administration regimen is necessary. Probably the nearest clinical application in the reperfusion injury is the use of sodium–hydrogen exchange (NHE) inhibitors [193]. These agents inhibit the Na1 / H1 exchange system, activation of which leads to Na 1 and Ca 21 overload (and hence cell death) during both ischemia and reperfusion. Cariporide, a potent inhibitor of the NHE-1 system has been extensively studied. A detailed review of its effects was published recently [194], but it is worth stressing that it reduced migration of neutrophils to the ischemic and reperfused tissue [195]. Rupprecht et al. studied the influence on infarct size and myocardial function of administration of intravenous cariporide just before the reperfusion, in patients with acute anterior myocardial infarction treated with the direct PTCA. They applied very rigorous selection criteria, enrolling only patients with a totally occluded left anterior descending artery who presented within 6 h of the onset of pain. Ventriculograms of

patients treated with the NHE inhibitor, recorded 3 weeks after PTCA, showed a better ejection fraction and fewer prominent wall motion abnormalities [196]. On the other hand, GUARDIAN, the first big clinical trial with cariporide, which enrolled 11590 patients at high risk of ischemic events (patients with unstable angina undergoing high risk revascularisation procedures) showed a relatively minor effect that was limited to patients receiving the highest dose [197]. Only patients undergoing bypass surgery benefited from this treatment. This study illustrates well how far it is from the laboratory bench to the bed of a patient, but NHE inhibitors remain a good prospect for future prevention or therapy in acute coronary syndromes. The importance of integrins CD11b / 18 in the process of activation of leukocytes makes them a plausible target for intervention. A cross-reactivity between b3-integrin antagonists (abciximab) and CD11b / 18 has been described [198]. Neumann et al. demonstrated that intravenous abciximab protects microvascular integrity after stent placement for myocardial infarction [199]. A recently published head-to-head comparison showed the superiority of abciximab over tirofiban (a non peptidic gpIIb / IIIa inhibitor) in the prevention of ischemic events after percutaneous interventions [200]. This difference may be partially explained by abciximab’s affinity to receptors other than those of a2b3 integrin. One of the interesting prospects for future therapy is inhibition of the terminal complement components. Anti-C5 therapy with monoclonal antibodies significantly reduces PMN infiltration, and most importantly reduces infarct size after both short and long periods of reperfusion. Antibodies against C5 inhibited both necrosis and apoptosis [201]. Other studies seem to confirm the involvement of complement activation in various clinical conditions where reperfusion takes place after a period of ischemia, such as PTCA in unstable angina [202] or operations with cardiopulmonary by-pass [203]. Inhibitors of C1-esterase were also reported to improve outcome after ischemia / reperfusion making them a novel investigation subject in this field. Another substance with possible therapeutic prospects in ischemia / reperfusion injury is acetaminophen. It is the most often used painkiller in western countries. This phenol with antioxidant properties

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also inhibits the function of neutrophils [204] and modulates the activities of myeloperoxidase [205]. In addition, in the experimental setting it protects the function and structure of ischemic / reperfused myocardium by attenuation of hydroxyl radical and peroxynitrate formation [206]. Yue et al. reported protective effect of PPARg agonist rosiglitazone of reperfused myocardium [207] Although the exact mechanism of this protection is unclear, inflammatory response inhibition, including cytokine release suppression, adhesion molecules and lymphocyte recruitment diminishing are considered to play a role. Genetic engineering is liable to enter cardiology clinics sooner than we once thought. Most likely it will also find its place also in the treatment of myocardial injury. So far the experiments suggest good prospects. Reduction of infarct size by delivery of the SOD [46] or eNOS [208] gene to myocardium was already mentioned. Nevertheless, new directions of research have been proposed. Heat shock proteins (HSPs) are group of agents that play a very important role in the cell’s resistance to stress stimuli. They associate with newly produced proteins that have not reached their permanent folding state, and prevent their denaturation. Recent studies show that transfection of heart cells with HSP70 gene reduces infarct size after reperfusion [209].

7. Perspectives for future study Despite impressing amount of data we are still far away form explaining all the mechanisms directing reperfusion injury. Almost every month bring new fascinating insights, which sometimes profoundly change our understanding. Just like the mentioned above experiment with p47 phox mice, devoid of NADPH oxidase, which present the same reperfusion injury as wild types [171]. This is just one of the examples which show how much genetic engineering can contribute to the cardiovascular research. Knockout mice lacking particular genes or their combinations as well as transgene animals overexpressing certain proteins will surely help to explain the sequence of key processes. In respect to this, genes coding elements of transduction pathways seem to be especially promising. Moreover unfolding in-

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volvement of particular components may create an incentive to produce selective acting drugs, maybe such a breakthrough like reperfusion therapy. Unfortunately not all genes can be knocked-out using conventional methods. It results sometimes in prenatal death. Another shortcoming of the conventional techniques is that all tissues bear the same mutations. But expression of certain genes yield different results depending on in which tissue expression takes place. iNOS is here a good example. In myocytes it takes a key position in preconditioning and cytoprotection whereas in macrophages is responsible for inflammation-related organ injury. A recent discovery of Cre-lox system enables to omit the perinatal mortality, and develop tissue specific knockouts [211]. Then excision of the crucial part of the gene takes place only in cells expressing specific proteins. For example coupling Cre with a a-myosin heavy chain (aMHC) promoter, results in cardiomyocyte-specific knockout which occurs in postnatal life. Similarly, coupling with a drug-inducible promoter allows to switch the knockout on later in life [212]. This is a wonderful tool allowing manipulating single genes in single cells. But on the other hand these new instruments require very precise planning and scrupulous implementation. Using knockout animals in different models, e.g., in vivo I / R, Lagendorf perfused heart or isolated cardiomyocytes often bring conflicting results. All they cannot fully answer the question what is the role of given gene in human heart during ischemia and reperfusion, but what is its role in the process analysed in this particular setting. After all no patient runs on the street with a conditional knockout in cardiomyocytes.

8. Conclusion Reactive oxygen species and neutrophils are the key players in reperfusion injury, interacting at multiple sites with each other, as well as with other elements such as the complement system, endothelial cells, lymphocytes, macrophages and myocytes. These overlapping activities make the activation cascades extremely redundant, and hence making inhibition of a process becomes much more complicated than the mere inhibition of a single receptor.

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Pleiotropic substances such as Na1 / H1 exchange inhibitors, adenosine or nitric oxide donors, acting at different levels on various cells, therefore have a better chance of obtaining a satisfactory effect. The era when cytokines were considered to be ‘‘good’’ or ‘‘bad’’ has gone by like the black-and-white westerns. Minor changes of concentration, receptor density or the presence of co-acting substances may frequently cause a dramatic change of outcome. It is worthy to keep in mind that reperfusion injury affects all organs, and is one of the main reasons of early organ failure after transplantation of a liver or a kidney. It still remains unclear why some patients recover from serious myocardial infarction quickly after reperfusion therapy, and with hardly any contractile dysfunction, while others, who on admission present with similar clinical status and are treated in the same way, develop heart failure and often die within days after apparently successful reperfusion. A better understanding of mechanisms underlying reperfusion injury will be the key for developing successful therapy attenuating the adverse consequences of acute coronary syndromes, coronary surgery and transplantations.

Acknowledgements The authors thank Ms. Michelle Withers MRCS and Professor Jan Dlugosz for their critical reading of the manuscript.

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