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MODULATION OF LEUKOCYTE-MEDIATED MYOCARDIAL REPERFUSION INJURY Benedict R. Lucchesi The University of Michigan Medical School, Department of Pharmacology, M6322 Medical Science Building I, Ann Arbor Michigan, 48109-0626 KEY WORDS:
neutrophils, free radical, ischemic myocardium, inflammatory mediators, chemotactic stimuli
INTRODUCTION New therapeutic interventions, such as coronary angioplasty or thrombolytic therapy, are directed towards the treatment of acute myocardial ischemia based upon the recognition that mortality among patients who are evolving a myocardial infarction is influenced by the extent of left ventricular dysfunc tion, which is directly related to the amount of myocardium that becomes infarcted and thus non-functional
(1).
Secondly, the size of the infarct that
results is related directly to the severity and duration of the ischemic interval. Patients who are treated and reperfused early
«4
hr) after the onset of
symptoms exhibit better functional recovery as measured by a larger ej ection fraction and smaller infarct sizes as assessed on the basis of myocardial intracellular enzyme release (2). Third, reperfusion of the acutely ischemic myocardium results in a reduction in ultimate infarct size as determined in the experimental animal and presumably in patients. Proper aggressive treatment of acute myocardial infarction should involve attempts to recannalize the obstructed coronary vessels either by mechanical means or by thrombolytic therapy
(3). 561
0066-4278/90/0315-0561$02.00
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The size of the myocardial infarct resulting from a period of regional ischemia is related directly to the severity and duration of the ischemic insult (4). In the canine heart, coronary artery occlusion for more than 4 hr followed by reperfusion results in an infarct that is essentially identical to the extent of infarction that results after permanent coronary artery occlusion for 24 hr. Coronary occlusion less than 20 min is associated with prolonged ventricular dysfunction; a phenomenon referred to as the stunned myocardium (5) that eventually recovers without evidence of myocardial cell injury. Inasmuch as the degree of myocardial cellular necrosis increases with increasing duration of ischemia, it is apparent that early reperfusion of the ischemic myocardium will result in salvage of potentially viable tissue (6). When the ischemic interval is extended to 40 min, the extent of irreversibly injured tissue (infarct size) is related directly to the duration of the ischemic event. Reimer & Jennings (4) described the "wavefront phenomenon" of advancing necrosis from the subendocardial to the subepicardial regions of the myocardium. The duration of protracted blood flow deprivation to the myocardium increased the extent of irreversibly injured myocardium and decreased the amount of tissue that could be salvaged by reperfusion. Most of the remaining viable tissue was confined to the subepicardial region, un doubtedly because of the maintenance of coronary blood flow provided by means of collateral vessels. It is clearly evident that early reperfusion of the ischemic cardiac muscle is essential if the goal is to prevent or reduce the extension of tissue damage associated with regional myocardial ischemia caused by a myocardial infarction. THE CONCEPT OF MYOCARDIAL REOXYGENATION INJURY Reperfusion of the ischemic myocardium results in an apparent acceleration of necrosis as manifested by cell swelling and the formation of contraction bands. This has been referred to as myocardial reperfusion injury. The sudden reintroduction of oxygenated whole blood reperfusion may be detrimental to the once ischemic myocyte, thereby giving rise to a paradoxical situation concerning the best approach to preserve the ischemic heart and the concept of myocardial reperfusion injury. Because the cellular morphologic changes observed with reperfusion are dependent upon the presence of molecular oxygen, it would be more appropriate to think in terms of reoxygenation injury (7). As a simple definition, myocardial reperfusion (reoxygenation) injury re fers to irreversible cellular damage (necrosis) resulting from the reintroduc tion of molecular oxygen at the time of organ reperfusion. This suggests that the reintroduction of oxygen itself causes injury that would not have occurred
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at all or at least not as rapidly as without perfusion. Injury to the myocardial cell upon whole blood reperfusion is characterized by a variety of events such as calcium overload into cells that have preexisting membrane defects, the influx of inflammatory cells (e.g. neutrophil), the generation of oxygen derived free-radicals or other long-lived oxidants and the release of proteoly tic enzymes associated with the acute inflammatory response. Reperfusion injury in vitro may be somewhat different under in vivo conditions, which involve the cumulative effects of one or more tissue or blood-borne factors. The alterations in myocardial cell viability observed under in vitro conditions may be a reflection of the intracellular generation of reactive species of oxygen and may differ significantly from what is observed in the intact heart perfused with oxygenated, whole blood. The tissue damage initiated with the reintroduction of whole blood will continue for an indeterminate period after
reperfusion has been established. The subsequent regional myocardial in flammatory response to tissue injury and the accumulation of polymorphonu clear leukocytes at the site of injury secondary to the damage inflicted upon the myocardium due to the earlier ischemic insult is responsible for the extension of the tissue damage that occurs with reperfusion (8-10). For the purpose of this presentation, we will employ a strict definition of reperfusion injury that considers the possibility that some or all of the ischemic myocytes that are not salvaged by reperfusion are viable at the time of reperfusion, but undergo a lethal explosive alteration because of an im posed oxidative stress that is associated with the reintroduction of oxygenated blood and the associated cellular components. Myocardial cells that were viable at the end of the ischemic interval are subjected to unfavorable con ditions related to reperfusion, and it is the act of reperfusion itself that leads to cell death. This is the basis for the concept of reperfusion injury as used in this review. Myocardial cell death due to reperfusion injury may be different with respect to the mechanism of death than from that which occurs from pro longed myocardial ischemia.
POTENTIAL SOURCES FOR THE PRODUCTION OF REACTIVE SPECIES OF OXYGEN
There is a complex interrelationship among the various factors that contribute to reperfusion injury. Oxygen-derived free-radicals are produced by activated neutrophils that infiltrate the ischemic and reperfused myocardium. Free radical scavengers as well as the oxygen-derived free-radical metabolizing enzyme, superoxide dismutase, and the hydrogen peroxide degrading en zyme, catalase, reduce the contribution to myocardial cell injury caused by superoxide anion and hydroxyl radical (II, 12). The polymorphonuclear
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neutrophil and other phagocytic cells possess the capacity to produce reactive species of oxygen when presented with appropriate stimuli
(13) in a metabolic
event that is referred to as the respiratory burst. The oxidants produced by the activated phagocytes consist of superoxide anion (027), hydrogen peroxide (H202), hypochlorous acid (HOCl), chloramines (RNHCl-), and hydroxyl anion (·OH). The primary function of these oxidants is to provide a defense
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mechanism against invading microorganisms. The killing of microorganisms occurs within the phagocytic vacuole in which oxidants are contained within an environment well-suited for such reactive products. The reactive species of oxygen become deleterious to surrounding cells when they are released into the extracellular environment. The primary reactant that serves as the ultimate source for each of the oxidants is 027, which results from the one-electron reduction of oxygen catalyzed by the enzyme NADPH-oxidase. The oxidase, a membrane-bound flavoprotein, which is dormant in the resting phagocytic cell, becomes activated when stimulated by components of the complement system (C5a). Neutrophils contain the enzyme myeloperoxidase, which catalyzes a H20r dependent oxidation of halide ions that involves both CI- and Br- ions to give rise to OCl- or OBr-. Each of these hypohalite anions can act as a powerful
oxidant capable of attacking a wide variety of biomolecules including
al
antiproteinase. An additional potential source of oxygen-derived free-radicals and oxidants is the enzyme, xanthine oxidase, which is localized within the vascular endothelium in many animal species studied to-date, but is not believed to be present in the myocyte of the human heart
(14). Xanthine dehydrogenase
undergoes a conversion from it s dehydrogenase to its oxidase form because of the activation of a calcium-dependent protease (14, 15). Upon reperfusion, xanthine oxidase is believed to utilize hypoxanthine as a substrate and molecular oxygen as an electron acceptor, thereby leading to the production
of superoxide anion (027). Support for this concept derives from the observa tion that allopurinol, an inhibitor of xanthine oxidase, has been reported to reduce tissue injury associated with myocardial reperfusion (15, 16). Studies that demonstrate the protective effects of allopurinol in ex perimental models of myocardial ischemia provide evidence that xanthine oxidase may participate in the generation of oxygen-derived free-radicals during myocardial reperfusion
(17). Under conditions of ischemia it appears
that the enzyme xanthine dehydrogenase is converted by a calcium-activated protease to the oxidase form, xanthine oxidase, (D to 0 conversion)
(18).
Additionally, during ischemia there is an accumulation of purine metabolites from ATP and ADP (AMP, inosine, hypoxanthine). Upon reperfusion, the required substrates hypoxanthine and the necessary electron acceptor oxygen, are provided to the enzyme, which leads to the formation of uric acid and the
REPERFUSION INJURY
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generation of superoxide anion. The localization of xanthine oxidase, primari ly in the endothelial cell 1 ( 4), which to induce tissue injury. Consistent with this premise is the reported observation that superoxide dismutase plus catalase administered one min before reperfusion reduced both the microvascular damage and the low reflow phenomenon in the endocardium of the canine heart subjected to regional
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ischemia for
2 hr followed by reperfusion for 4 hr (19).
Kehrer et al (20) were unable to detect any significant degree of conversion of xanthine dehydrogenase to xanthine oxidase in the rat heart, and pretreat ment with allopurinol did not provide protection against the release of in tracellular enzymes when the hearts were reoxygenated after a period of hypoxia. It was concluded that the tissue injury in the hypoxic reoxygenated
rat heart was the result of a mechanism other than reactive oxygen generated et al (21),
by xanthine oxidase. A similar view was expressed by Bindoli
which indicated that the cardioprotective effect of allopurinol in the rat should be referred to as a mechanism that is unrelated to one based upon the inhibition of xanthine oxidase. Allopurinol does not prevent the formation of superoxide anion despite the fact that it can inhibit the production of uric acid by xanthine oxidase as opposed to its active metabolite oxypurinol, which can inhibit both functions of the enzyme (22). Some degree of caution is needed in interpreting data derived with allopurinol. The drug can not be regarded, when used in vivo, as a specific inhibitor of xanthine oxidase. According to more recent studies
(16), it was suggested that metabolic conversion of
allopurinol to oxypurinol may permit the active metabolite to exert an action to counteract the interaction of neutrophils with the coronary vasculature and/or with the cardiac myocytes.
THE ROLE OF REACTIVE OXYGEN METABOLITES IN REPERFUSION INJURY The first suggestive evidence of the participation of oxygen-derived free radicals in myocardial reperfusion injury in the intact animal developed from the work of Jolly et al 1 ( 1). tration of the superoxide anion metabolizing enzyme, superoxide dismutase (SOD), and the H202 degrading enzyme, catalase, to anesthetized dogs subjected to 90 min of coronary artery occlusion followed by reperfusion was effective in reducing ultimate infarct size. In contrast, the antioxidant en
40 min after reperfusion had begun. (23) subsequently showed that the administration of SOD (5 mg/kg; 3800 U/mg) alone resulted in a 50% reduction in myocardial infarct
zymes were without benefit if infused Wems et aI
size, whereas the administration of catalase alone was not accompanied with a significant degree of myocardial salvage. Ambrosio et al
(24) demonstrated
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LUCCHESI
that recombinant human superoxide dismutase could reduce ultimate infarct size in the canine heart subjected to
90 min of regional ischemia followed by
reperfusion, despite the fact that the enzyme was administered immediately upon reperfusion. The latter observation agrees with other similarly con ducted studies
(8, 11,12,25) and supports the contention that an extension of
myocardial injury occurs coincident with the onset of reperfusion. It is of
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importance to note that not all studies are in agreement. The administration of superoxide dismutase plus catalase to the canine heart subjected to either 40 (26) or 90 (27) min ischemia followed by reperfusion for 4 days failed to provide evidence for a protective effect. Several investigators who have employed the chronically instrumented canine heart also have failed to observe a reduction in ultimate infarct size with native superoxide dismutase
(28, 29) given in combination with catalase. & colleagues (28) employed a 3 hr period of ischemia followed by reperfusion for 24 hr, whereas Nejima et al (29) used a 90 min period of regional ischemia followed by 7 days of reperfusion. One biological variable that could account for the differing results among Gallagher
the various studies with SOD is the duration of occlusion used to induce the infarcts. Studies reporting positive results employed durations of occlusion of
60 or 90 min. Those reporting negative results have used ischemic intervals of 40 min or 3 hr. Reperfusion injury may require an ischemic period of greater than 40 min and less than 3 hr. The failure to substantiate a protective effect in the above mentioned studies may be model dependent. The degree of myocar dial cell death associated with a
3 hr ischemic interval may have resulted in far
too little myocardium to be salvaged as a result of the applied intervention
(30, 31). On the other hand, it is possible that an ischemic period of 40 min may be insufficient for the demonstration of myocardial injury due to a neutrophil-dependent, oxygen-radical-mediated mechanism. Eng et al
(32) provide an interesting insight into the relevance of the
duration of coronary artery occlusion in studies designed to assess in terventions for the protection of the ischemic heart. The latter investigators demonstrated that there is an explosive increase in the extent of necrosis between a narrow 50 to
60 min period of ischemia. Approximately 70% of the 90 min of severe blood flow
region at risk became necrotic when subjected to
deprivation, whereas only a limited extent of myocardial injury (approximate ly
10% of the risk region) was observed with a severe deprivation of coronary 40 min. Short periods of ischemia (e.g. 40 min) may
blood flow that lasted for
not be able to elicit a component of injury because of reperfusion, and most of the detected cellular damage is related to myocardial metabolism. Ischemia itself and the metabolic demands of the tissue at the time become the primary determinants of cell viability within the ftrst detected as a result of
20 min. Necrosis was not 20 min of severe ischemia. Beginning at 30 min of
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blood flow deprivation, necrosis progressed from the endocardium toward the epicardium in a wave front pattern so that 90 min of ischemia resulted in 70% of the risk region undergoing irreversible injury. There is support for the concept that myocardial ischemia and, in particular reperfusion injury, are accompanied by the formation of oxygen-derived free-radicals, which overwhelm diminished endogenous protective mech anisms to exacerbate cell injury. Cell death is probably a far more violent event than is often imagined, "an explosion rather than a dissolution", since the generation of free-radicals occurs rapidly, and the half-life of the reactive oxygen species, in most cases, is of relatively short duration. Recent studies utilizing electron spin resonance spectroscopy and spin trapping agents have provided direct evidence for the generation of oxygen-free radicals in myocar dial tissue during the ischemic interval and particularly during reperfusion (33-35). Short durations of ischemia are not associated with irreversible cellular changes. The detection of free-radical production became apparent during the time of coronary occlusion and increased 100-fold during reperfu sion (36). The burst of free-radical production peaked at 2� min after reperfusion and persisted for 3 hr after reflow. Relatively short periods of regional ischemia are accompanied by free-radical generation at the time of flow deprivation and by an explosive and prolonged period of free-radical production well into the period of reperfusion. The formation of both oxygen centered and carbon-centered radical species appears to be a consequence of oxy-radical attack of cardiac membrane lipids and supports the idea of lipid peroxidation in the pathogenesis of ischemic heart damage, particularly dur ing reperfusion (35). Free-radical production by endothelial cells, along with an inhibition of oxy-radical production by oxypurinol, provides additional support for the potential role of reactive oxygen products in ischemia and reperfusion injury (37). The sustained generation of cytotoxic oxygen radicals (36) may explain why interventions such as superoxide dismutase with its relatively short pharmacologic half-life of 6--10 min may appear to be ineffective in ex perimental protocols designed to assess tissue damage 4 or more days after reperfusion (26, 39, 29). The scavenging action of the enzyme would have dissipated because of its short half-life, at a time when there is a continued production of lipid peroxidation because of oxygen-free radical attack on biological membranes. Przyklenk & Kloner (41) demonstrated significant protection by superoxide dismutase in the heart subjected to 6 hr of regional ischemia without reperfusion. If perfusion was instituted, the protective effect of the scavenger was no longer apparent at 30-48 hr post reperfusion. It was concluded that superoxide dismutase delayed rather than prevented the de velopment of cell death. That this reasoning may be incorrect was suggested by a subsequent study in which, polyethylene glycol conjugated superoxide
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dismutase, with a pharmacologic half-life of ca 30 hr, not only reduced infarct size in the heart subjected to 6 hr of regional ischemia, but continued to protect against cell death when infarct size was assessed 30 hr after reperfu sion (42).
ROLE OF NEUTROPHILS AS A SOURCE OF REACTIVE OXYGEN METABOLITES AND PROTEOLYTIC ENZYMES AS POTENTIAL MEDIATORS OF MYOCARDIAL CELL INJURY Neutrophils are capable of being activated upon attachment to the vascular endothelium with the subsequent generation of toxic oxygen products and the release of destructive proteases capable of altering vascular permeability. Acid and neutral proteases are also released that can alter the integrity of the vascular wall basement membrane (43). The neutrophil contains two latent metalloproteinases, collagenase and gelatinase, that are activated by hypochlorous acid and are capable of degrading collagen in addition to lysing endothelial cells (44). Mediators present during myocardial ischemia capable of activating neutrophils include complement activation products, leukotriene B4, and platelet activating factor. It was suggested that activation of neutrophils within the vascular space results in the formation of cellular aggregates that can physically impair blood flow to the myocardial capillary bed and thereby exacerbate the ischemic injury (45). Neutrophil accumulation in the vascular bed is thought to be the reason there is sometimes inadequate reflow or the no-reflow phenomenon after brief periods of ischemia (46) followed by reperfusion. Associated with reperfusion of the ischemic myocardium is a cellular reaction characterized by the accumulation of inflammatory cells and the production of inflammatory mediators in the reperfused myocardium. The regional accumulation of inflammatory cells contributes, in part, to the extension of tissue injury associated with reperfusion of the previously ischemic myocardium (47-49). Leukocyte/endothelial cell interaction is es sential for leukocyte migration across the endothelial surface, and superoxide anion, as well as C5a, have been demonstrated to increase adhesion of neutrophils to the endothelium (50). Several studies have documented the correlation between infarct size result ing with reperfusion and the extent of neutrophil infiltration (48, 51, 52). Neutrophils have been observed to infiltrate the damaged myocardial region beginning with the onset of ischemic injury and to increase their numbers progressively for the first 24 hr post myocardial infarction (53). The infiltra tion of neutrophils within the early postischemic interval before the develop ment of myocardial necrosis has been demonstrated (54-55). There is a
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direct relationship between the duration of myocardial ischemia and the extent of neutrophil infiltration and accumulation in the reperfused region (56). The formation of tissue edema, which accompanies cellular injury, involves in teractions dependent upon the neutrophil for the full expression of the in tlammatory response (57). The generation of oxygen-derived free-radicals represents one of the main mechanisms by which polymorphonuclear neutro phils can mediate tissue injury. The reactive products of oxygen are formed during the engulfment of particulate matter or when the neutropbils are activated by soluble stimuli, particularly C5a. Romson et al (52) demonstrated that the myocardial protective effect of the non-steroidal anti-inflammatory agent, ibuprofen, was related to the inhibi tion of neutrophil accumulation in the reperfused myocardium or risk region. Evidently ibuprofen reduced myocardial infarct size in the dog subjected to 60 minutes of coronary occlusion followed by 24 hr of reperfusion. The anti inflammatory effects of ibuprofen resulted in decreased neutrophil accumula tion in the myocardial risk region witb a concomitant salvage of tissue in the area subjected to reperfusion. The protective effects of ibuprofen are un related to its ability to inhibit cyc100xygenase since neither acetylsalicylic acid nor indomethacin reduced ultimate infarct size or inhibited the release of superoxide anion by activated neutrophils (58). Ibuprofen, in contrast to indomethacin and acetylsalicylic acid, provides a protective effect via a pharmacologic mechanism that does not involve its ability to inhibit the formation of prostanoids and most likely is acting by a direct action upon the intlammatory cells by impairing their cbemoattraction to the region and impairing their ability to form and release cytotoxic derivatives. Pharmacologic interventions capable of inhibiting the lipoxygenase path way of arachidonic acid metabolism also reduce myocardial infarct size probably through a mechanism related to the inhibition of neutrophil accumulation within the myocardium. The dual cyclooxygenase-lipoxygenase inhibitor BW 755C (10 mg/kg) reduced myocardial infarct size by 50% as compared to non-treated controls when evaluated with a canine model of 90 min of coronary occlusion followed by 24 hr or reperfusion (55, 59) in association with a reduction in neutrophil accumulation in the reperfused myocardium (55),
CHEMOTACTIC STIMULI AND ACTIVATION OF NEUTROPHILS In order for neutrophils to injure ischemic tissue, there must be a chemical signal or chemoattractant to direct the neutrophil to the affected myocardial region. Ischemic myocardial tissue gives rise to a tissue protease that activates the third component of complement (60). Extensive confirmation dem-
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onstrates that myocardial ischemia activates the complement system and that ischemia results in the migration of neutrophils into the jeopardized myocar dium (61-63). Other chemotactic factors are generated in response to myocar dial tissue injury, including leukotriene B4. A chemotactic factor can be produced by a superoxide anion acting upon a plasma protein that has been characterized as a derivative of arachidonic acid (64). An increase in chemotactic activity is noted in the coronary sinus blood of dogs subjected to ligation of the left anterior descending coronary artery and correlates with the enzymatic and electrocardiographic indices of myocardial ischemia (65). Additional evidence for the participation of the complement system in the development of myocardial ischemic injury is obtained from studies in which complement depletion with cobra venom factor after coronary ligation re sulted in a reduction in the amount of myocardium that eventually became infarcted (66). These observations were confirmed with the use of im munohistochemical methods demonstrating an extensive localization of com plement components (C3, C4, and CS) within the ischemic myocardium which did not occur after decomplementation of the animal (61, 62). The membrane attack complex (MAC or attack sequence) of complement (C5b-9) is present within the ischemic myocardium of acutely infarcted humans as early as 6 hr after the onset of symptoms (67). The membrane attack complex consists of a stable macromolecular complex (C5b and C6, C7, and C8) that inserts into the membrane bilayer (with C9) and forms a pore or channel through the membrane that permits bidirectional flow of ions and macro molecules ultimately resulting in cell lysis. Thus, complement may have a direct as well as an indirect (neutrophil) effect on the sequential events associated with myocardial tissue injury and subsequent cell death. THE ROLE OF THE ENDOTHELIAL CELL
A pathophysiologic finding in myocardial ischemic injury is the presence of endothelial damage to the coronary vascular bed, which leads to the ex travasation of plasma and cellular components into the interstitial space. Polymorphonuclear leukocytes can adhere and migrate through the endothe lial cell layer and are of pathophysiologic relevance with respect to their potential role in mediating myocardial injury. This latter event is a time dependent process in the presence of total vessel occlusion. The inflammatory cells enter the damaged myocardium slowly and become apparent at 24 hr after the onset of injury. On the other hand, reperfusion of the once ischemic tissue allows for rapid access of the inflammatory cells to the jeopardized myocardial region (68). The movement of neutrophils and subsequent release of proteinases across the vascular wall may in itself be associated with permeability changes and injury to the vessel. The migration of the neutrophil
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across the connective tissue barrier of the blood vessel is dependent upon the action of proteolytic enzymes, which remain active even in the presence of plasma antiproteases (69). The adhesion of neutrophils to endothelial cells in culture is stimulated with endotoxin, interleukin-l (lL-l), or tumor necrosis factor, which suggests that the endothelial cell plays an important role in regulating the entry of the inflammatory cells to sites of tissue injury. Be vilaqua & co-workers (70) have shown that there are subtle and profound interactions between leukocytes and the vascular endothelium that are under the influence of immune modulation. It was suggested that IL-I can activate the vascular endothelium to increase the expression of its adherence mole cules for leukocytes (71). These molecules are distinct from the CDw18, LFA (Mol, gp150,95) class of glycoproteins and appear to be independent of the leukocyte receptors for the formyl peptides such as f-Met-Leu-Phe or the complement receptors for C3b or C5a. ADHERENCE PROMOTING CELL SURFACE GLYCOPROTEIN COMPLEXES A group of neutrophil cell-surface glycoproteins mediates cell-cell in teractions (72). The heterodimeric glycoprotein referred to as Mol and Mac-l antigen consists of an alpha chain, CD lIb (165 kd); and a beta subunit, CD 18 (96 kd). The members of the leukocyte family each possess the same beta subunit that is covalently linked to the alpha subunit. The latter will vary, thereby giving rise to a family of membrane glycoproteins, LFA-I, Mol and gp150,95 (73). The alpha subunits dictate the molecular specificity. The expression of these molecules on the cell surface will be determined by the origin and stage of maturation of the leukocytes (74). Activation of leukocytes by con A, FMLP, zymosan-activated serum, C5a, or calcium ionophore results in an enhanced expression of cell-surface adhesion glycoprotein mole cules in which upregulation is the result of a translocation of preformed receptors to the plasma membrane from an intracellular site. The process of expression on the cell surface is rapid and is not dependent upon new protein synthesis, which results in a 5-1O-fold increase in the number of glycoprotein receptors from the resting state of 10,000 to 20,000 molecules per cell (75, 76). Pathophysiologic conditions occur in which there is a chemoattraction and an enhanced binding of neutrophils to the surface of the affected cells. The localization of complement components may be a prerequisite for the attach ment of neutrophils to the vascular endothelium after an ischemic insult to the tissue. Mol receptors on the neutrophil bind C3bi and thus promote the adherence to cells coated with C3bi. If neutrophil adhesion to the target tissue is prevented with an anti-Mol monoclonal antibody, there is an associated
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reduction in the extent of tissue injury
(77). Neutrophil activation is accom
panied by the expression of the adhesion promoting receptors, the net result of which is an amplification of the process of neutrophil accumulation at the endothelial surface.
The resulting microenvironment at the neutrophil
endothelial cell-cell interface becomes the site at which the cytotoxic in flammatory mediators can exert their effects uninhibited by blood transported
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antioxidants and other cytoprotective factors.
THE INHIBITION OF NEUTROPHIL ADHESION 45 min after the induction of region al myocardial ischemia results in a significant protection against reperfusion injury as measured by the size of the myocardial infarct that results after 6 hr of reperfusion (78). There was no effect of the antibody on arterial blood
Administration of the anti-Mo l antibody
pressure, heart rate, or coronary artery blood flow that could account for the protective action. Anti-Mol administration in vivo significantly reduced neu trophil accumulation within the ischemic myocardial tissue as assessed by histological analysis of tissue sections. The specificity of the monoclonal antibody for the Mol receptor provides supportive evidence that the infiltrat ing neutrophil is a major contributor to the observed extension of myocardial injury at the time of reperfusion.
CONCLUSIONS It is now accepted that early reperfusion of the once ischemic myocardium is an important consideration in an effort to reduce overall loss of functional tissue within the region of myocardium at risk. Thus reperfusion is essential for survival of the jeopardized heart muscle, and the reduction in ultimate infarct size is associated with a decrease in morbidity and mortality. There will exist within the myocardial region at risk, a population of myocardial cells that are reversibly injured as a consequence of a period of (less than 3 hr) of ischemia. Within this population of cells, a fraction of the affected myocytes might undergo irreversible changes (cell death) associated with the onset of reperfusion. Reperfusion injury, therefore, refers to cell death caused by reperfusion. It is suggested that the lethal event may be reduced or prevented by appropriate measures directed against the cytotoxic effects of reactive species of oxygen and perhaps proteolytic products derived from inflammatory cells that are attracted to the injured area as a result of the influence of complement- and arachidonic-acid-derived chemoattractants, in terleukin-l, and adhesion promoting neutrophil glycoproteins. Among the sites of production of the oxygen-derived free-radicals, one might include phagocytic cells, myocardial tissue, especially the mitochondria of the reper-
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fused myocyte, and the vascular endothelial cells that possess the enzyme xanthine oxidase. Both oxygen metabolite-dependent and -independent pro cesses may participate in the explosive tissue injury that is associated with the onset of reperfusion and additive to the myocardial damage that is secondary to the ischemic insult itself. The two events may be attributed to entirely different mechanisms of cell injury. This view may be at odds with the observations reported by others (79), and additional effort must be devoted towards a better understanding of the role of the inflammatory mediators that are attracted to the myocardial risk region within moments of the onset of reperfusion. ACKANOWLEDGMENTS
The original work reported from the author's laboratory was supported by a grant from the National Institutes of Health, Heart, Lung and Blood Institute, HL-19782-1O. The author acknowledges the valuable contributions of his colleagues Dr. Paul Simpson, Dr. Steven Werns, Dr. Robert Todd III, and Dr. Joseph Fantone. Literature Cited I. Rude . R. E Muller. 1. E BraunwaJd. E. 198 I. Efforts to limit the size of myocardial infarcts. Ann. Intern. Med. 95:736-61 2. Schwarz, F., Schuler, G., Katus. H., .•
.•
Hofman, M., Manthey, J., ct al. 1982. Intracoronary thrombolysis in acute myocardial infarction: Duration of isch emia as a major determinant of l ate re sults after recanalization. Am. J. Car dial. 50:933-37 3. Braunwald, E. 1985. The aggressive treatment of acute myocardial infarction. Circulation 71:1087-1092 4. Reimer, K., Jennings, R. 1979. The "wave-front phenomenon" of ischemic c ell death. n. Transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral blood flow. Lab. Invest.
40:663-44 5. Heyndrickx. G. R., M il lard. R. W.,
6.
�
McRitchie, R. J., Ma roko . P. R., Vat ncr, S. F. 1975. Regional myocardial functional and electrophysiological al terations after brief coronary artery occlusion in conscious dogs. J. Clin. Invest. 56:978-85 Koren, G., Weiss, A. T .• Hasin, Y .• Appelbaum, D., Wellber, S., et al. 1985. Prevention of myocardial damage in acute myocardial ischemia by early treatment with intravenous streptokinase. New Engl. J. Med. 313:1384--89
7. Hearse. D. J .• Hu mphrey . S. M .• Nay ler. W. G., Slade, A., Bordu, D. 1975. Ultrastructural damage associated with reoxygenation of the anoxic myocar dium. J. Mol. Cell. Cardiol. 7:315-24 8. Tamura, Y., Chi, L., Driscoll, E. M., Hoff, P. T., Freeman, B. A., et al. 1988. Superoxide dismutase conjugated to polyethylene g ly col provides sus tained pr otect ion against myocardial ischemialreperfusion injury in the canine heart. Cire. Res. 63:944--59 9. Simpson, P. J., Fantone, J. C., Luc chesi, B. R. 1988. Myocardial ischemia and reperfusion injury: Oxygen radicals and the role of the neutrophil. In Oxygen Injury, ed. B. Hal liwell. pp. 63--77. BetheSda, Md: Fed. Am. Soc. Exp. BioI. 10. Jacob, H. S., Vercellotti. G. M. 1988. Granulocyte mediated endothelial in jury: Oxidant damage amplified by lac torferrln and platelet activating factor. In Oxygen Radicals and Tissue Injury, ed. B. Halliwell, pp. 57--62. Bethesda, Md: Fed. Am. Soc. Exp. BioI. 11. Jolly, S. R., Kane, W. J., B ai lie , M. B., Abrams, G. D., Lucchesi, B. R. 1984. Canine myocardial reperfusion injury: Its reduction by the combined adminis tration of superoxide dismutase and cata lase. Res. 54:277-85 12. Werns. S. W., Simpson, P. J., Mickel son , J. K . , Shea, M. J .• Pitt, B., Luc-
Radicals and Tissue
Cire.
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chesi, B. R. 1988. Sustained limitation by superoxide dismutase of canine myocardial injury due to regional ische mia followed by reperfusion. J. Car diovasc. Pharmacol. 11:36-44 13. Babi or, B. M. 1978. Oxygen dependent microbial killing by phagocytes. New Engl. J. Med. 298:659-68 14. Jara sch, E.-D., Bruder, G., Heid, H. W. 1986. Significanc e of xanthine ox idase in capillary endothelial cells. Acta Physiol. Scand. 548(SuppL):39-46 15. Parks, D. A., Granger, D. N. 1986. Xanthine oxidase: Biochemistry, dis tribution and physiology. Acta Physiol. Scand. 548(SuppL):87-99 16. Werns, S. W., Shea, M. J., Mitsos, S. E., Dysko, R. C, Fantone, J. C. , et al. 1986. Reduction of the size of infarc tion by allopurinol in the ischemic reperfused cani ne heart. Circulation 73:518--2 4 17. DeWall, R. A., Vasko, K., Stanley, E., Kezdi, P. 1971. Responses of the ischemic myocardium to allopurinol. Am. Heart. J. 82:362-70 18. Battclli, M. G. 1980. Enzymic conver sion of rat liver xanthine oxidase from dehydroge nase (D-form) to oxidase (0fonn). FEBS Lett. 113:47-51 19. Przyklenk, K. , Kloner, R. A. 1989. R eperfusion injury" by oxygen-derived free radicals ? Effect of superoxide dis mutase plus catalase, given at the time of reperfusion, on myocardial infarct size, contractile function, coronary microvas culature and regional myocardial blood flow. Circ. Res. 64:86-96 20. K ehrer, J. P., Piper, H. M., Sies, H. 1987. Xanthine oxidase is not respons ible for reoxygenation injury in isolated perfused rat heart. Free Radic. Res. Commun. 3:69-71 21. Bindoli, A., Cavallini, L., Rigobello, M. P., Coassin, M., Di Lisa, F. 1988. Modification of the xanthine-converting enzyme of perfused rat heart during ischemia and oxidative stress. J. Free Radie. Bioi. Med. 4:163--67 22. S p ec tor, T. 1988. Oxypurinul as an in hibitor of xanthine oxidase-catalyzed production of su pero xi de radical. Bio ehem. Pharmacal. 37:349--52 23. Werns, S. W., Shea, M. J., Driscoll, E. M., Cohen, C., Abrams, G. D., et aL 1985. The independent effects of oxygen radical scavengers on canine infarct size. Reduction by super oxi de dismutase and not catalase. Cire. Res. 56:895-98 24. Ambrosio, G . , Becker, L. C., Hutchins, G. M., Weisman, H. F., Wei sfel dt, M. L. 1 986. Reduction in experimental in farct size by recombinant human super-
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-
"
oxide dismutase: Insights into the pathophysiology of reperfus i on injury. Circulation 74:1424-33 25. Tamura, Y., Saito, S., Hatano, M., Lucchesi, B. R. 1989. Limitation of myocardia l infarct size by polyethylene glycol-conjugated superoxide dismutase in a canine model of 90 min coronary occlusion followed by 4 day s of reperfu sion. Kokyu To Junkan 37:201-8 26. Uraizee, A., Reimer, K. A., Murry, C. E., Jennings, R. B. 1987. Failure of superoxi de dismutase to limit myocar dial infarct size in a 40 minute ischemia! four days reperfusion model in dogs. Circulation 75: 1237-48 27. Richar d V. J., Murry, C. E., Jennings, R. B. , Reimer, K. A. 1988. Therapy to reduce free radicals during early reperfu sion does not limit the size of myocardial infarcts caused by 90 minu tes of i sch emia in dogs Circulation 78:473--80 28. Gallagher, K. P., Buda, A. J., Pace, D., Gerren, R. A. , Shlafer, M. 1986. Fail ure of superoxi de dismutase and catalase to alter size of infarction in dogs after 3 hours of oc clusio n followed by reperfu sion . Circulation 73:1065-1076 29. Nejima, J. , Canfiel d, D. R., Manders, W. T., Knight, D. R., Cohen, M. V., et al. 1989. Failure of superoxide dis mutase and catalase to alter the size of infarcts and functional recovery in con scious dogs with reperfusion. Circula tion 78:143--5 3 30. Jolly, S. R., Kane, W. J. , Hook, B. G., Abrams G. D., Kunkel, S. L., Luc chesi, B. R. 1986. Reductionofmyocar dial infarct size by neutrop hil depletion: Effect of duration of occlusion. Am. Heart J. 112:682-90 31. Reimer, K. A. , Jennings, R. B . , Cobb, F. R., Murdock, R. R., Greenfield, J. C., et al. 1985. Animal models for pro tecting the ischemic myocardium: Re sults of the NHLBI cooperative study. Cire. Res. 56:651-65 32. Eng, C. , Cho, S . , Factor, S. M., Ki rk E. S. 1987. A nonflow basis for the vulnerability of the subendocardium. J. Am. Call. Cardiol. 9:374-79 33. Bl asig, 1. E., Ebert, B., Lowe, H. 1986. Identification of free radicals trapped d uring myocardial ischemia in vitro by ESR. Stud. Biophys. 116:35-42 34. Zweier, J. L., Flaherty, 1. T., Weis feldt, M. L. 1987. Direct measurement of free radicals generated foll owi ng reperfusion o f isc hemic myocardium. Proc. Natl. Acad. Sci. USA 84:1404-1407 35. Arroyo, C. M., Kramer, J. R., Leiboff, R. H., Mergner G. W., Dickens, B. F., ,
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,
,
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REPERFUSION INJURY Weglicki, W. B. 1987. Spin trapping of oxygen and carbon-centered free radi cals in ischemic canine myocardium. J. Free Radie. Bioi. Med. 3:3 1 3- 16 36. Bolli, R. 1988. Oxygen-derived free radicals and postischemic myocardial dysfunction ("Stunned Myocardium").
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G. A. 1988. Measurement of endothelial c ell free radical generation : Evidence for a central mechanism of free radical in jury in post ischemic tissue. Proc. Natl. Acad. Sci. USA 85:4046-4050 Deleted in proof Richard, V. J., Murry, C. E . , Jennings , R. B . , Reimer, K. A. 1988. Therapy to reduce free radicals during early reperfu sion does not limit the size of myocardial infarcts caused by 90 minutes of isch emia in dogs. Circulation 78:473--80 Deleted in proof Przyklenk, K . , Kloner, R . A . 1987.
Effect of oxygen-derived free radical scavengers on infarct size following six hours of permanent coronary artery occlusion: S alvage or delay of myocyte
necrosis? Basic Res. Cardiol. 82: 1 46-58 42. Chi, L., Tamura, Y . , Hoff, P. T . , M acha , M . , G allagher , K. P. , et a!. 1 989. The effect of superoxide dis mutase on myocardial infarct size in the canine heart after 6 hours of regional ischemia and reperfusion. A demonstra tion of myocardial salvage. Circ. Res. 64:665-75 43. Janoff, A . , Zelig, J. D. 1 968. Vascular injury and lysis of basement membrane in vitro by neutral protease of human leukocytes . Science 16:702-4 44 . S medly , L. A. , Tonnesen, M. G . , Has lett , c . , Guthrie, L. A . , Johnston, R. B . Jr. et al. 1 986. Neutrophil-mediated in jury to endothelial cells. Enhancement
by endotoxin and essential role of neu
trophil elastase. J. Clin. Invest. 77: 1233--43 45. Jacob, H. 1 98 1 . The role of activated complement and granulocytes in shock states and myocardial infarction. J. Lab. CUn. Med. 98:645-53 46. Engler, R . , Covell, J. W. 1987. Granu
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locytes cause reperfusion ventricular dysfunction after I S-minute ischemia in the dog . Cire. Res. 6 1 :2(}-'28 Lucchesi , B. R . , Mullane, K. M. 1986. Leukocytes and ischemia-induced myo cardial injury . Annu. Rev. Pharmacal. Toxicol. 26:20 1-24
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Effect of duration of occlusion. Am. Heart. J. l l2:682-9O 49. Hess , M. L . , Rowe , G . T., Caplan, M . , Romson, J . L . , Lucchesi, B . 1985. Identification of hydrogen peroxide and hydroxyl radicals as mediators of leuko cyte-induced myocardial dysfunction. Limitation of infarct size with neutrophil inhibition and depletion . Adv. Myocar diol. 5 : 1 59-75 50. Craddock, P. R . , Hammerschmidt, D. E . , Moldow, C. F . , Yamada , 0 . , Jacob, H. S. 1 979. Granulocyte aggregation as a manifestation of membrane interac tions with complement : Possible role in leukocyte margination, microvascular occlusion, and endothelial damage. Semin. Hematol. 16: 14-147 5 1 . Romson, J. L . , Hook, B. G . , Kunkel, S . L . , Abrams, G . D . , Schork , M . A . , Lucchesi, B . R. 1983. Reduction o f the extent of ischemic myocardial injury by neutrophil depletion in the dog. Circula tion 67: 1 0 1 6- 1 023 52. Romson, J. L . , Hook, B . G . , Rigot , V. H . , Schork , M . A., Swanson, D. P. , Lucchesi, B. R. 1982. The effect of ibuprofen on accumulation of I l l-indi um labeled platelets and leukocytes in experi mental myocardial infarction.
Circulation 66: 1 002-10 1 1 53. Fishbein, M . c., Maclean, D . , Maroko, P . R. 1 978. Histopathologic evolution of myocardial infarction. Chest 73:843-49 54. Engler , R. L . , Dahlgren, M. D" Peter son, M. A . , Dobbs, A . , Schmid Schonbein, G. W. 1986. Accumulation of polymorphonuclear leukocytes during 3-h experimental myocardial ischemia. Am. J. Physiol. 25 1 :H93-H IOO 55. Mullane, K . M . , Read, N., Salmon, J . A., Moncada , S . 1 984. Role of leuko cytes in acute myocardial infarction in anesthetized dogs: Relationship to myo
cardial salvage by antiinflammatory drugs. 1. Pharmacal. Exp . Ther.
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228:5 1 0-22 Go, L. 0 . , Murry, C. E. , Richard, V . J . , Weischedel, G . R . , Jennings, R. B . , Rei mer, K . A . 1988. Myocardial neutro phil accumulation during reperfusion
and reversible or irreversible ischemic injury. Am. J. Physiol . 255:H1 l88-
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V . , Williams, T. J. 1 98 1 . Control of vascular permeability by polymorphonuclear leukocytes in in flammation. Nature 289:64�50 Flynn , P. J . , Becker, W. K . , Ver cellotti, G. M . , Weisdorf, D. J . , Crad dock, P. R . , et al. 1984. Ibuprofen in hibits granulocyte responses to in flamm atory mediators: A proposed
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mechanism for reduction of ex perimental myocardial infarct size. In flammation 8:33-44 59. Jolly , S. R., Lucchesi, B. R. 1 983 . Effects of BW 755C in an occlusion reperfusion model of myocardial inj ury. Am. Heart J. 106:8- 1 3 60. Giclas , P. C . , Pinckard, R. N . , Olson, M. S. 1979. In vitro activation of com p lement by isolated heart subcellular membranes. J. Immunol. 122: 146-5 1 6 1 . McManus, L. M . , Kolb, W. P . , Craw ford, M. H . , O'Rourke, R. A . , Grover, F. L., Pinckard, R. N . 1983. Comple ment localization in ischemic baboon myocardium . Lab. Invest. 48:436-77 62. Pinckard, R. N . , O'Rourke, R. A . , Crawford, M. H. 1983. Complement localisation and mediation of ischemic injury in baboon myocardium . J. Clin . Invest. 66: 1050-1056 63 . Rossen, R. D . , Michael , L. H . , Kagiyama, A . , Savage, H. H . , Hanson, G . , et al. 1988. Mechanism of comple ment activation after coronary artery occlusion: Evidence that myocardial ischemia in dogs causes release of con stituents of myocardial subcellular origin that comple x with human C l q in vivo. Cire. Res. 62:572-84 64. Perez, H. D . , Goldstein, J. M. 1 980. Generation of a chemotactic lipid from arachidonic acid by exposure to a super oxide generating system. Fed. Proe. 39: 1 1 70 (Abstr.) 65. Hartmann, J. R . , Robinson , J. A . , Gun nar, R. M. 1 977 . Chemotactic activity in the coronary sinus after experimental myocardial infarction: Effects of phar macologic interventions on ischemic in jury. Am. J. Cardiol. 40:550-55 66. Maroko, P. R . , Carpenter, C. B . , Chiariello, M . , Fishbein, M. C . , Rad vany, P., et al. 1978. Reduction by co bra venom factor of myocardial necrosis after coronary artery occlusion . J. Clin . Invest. 6 1 :66 1-70 67. Mathey , D. G . , Schafer, H . , Schofer, J., Kruger, W . , Langes, K . , Bhakdi, S. 1986. Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium. Circulation 74(Suppl . II):372 (Abstr.) 68. Mullane, K . M . , Read, N . , Salmon, J. A . , Moncada, S. 1984. Role of leuko cytes in acute myocardial infarction in anesthetized dogs: Relationship to myocardial salvage by anti- inflamma tory drugs. J. Pharmacol. Exp. Ther. 228:5 1 0-22 69. Russo, R. G . , Liotta, L. A . , Thorgeirs-
son, U., B rundage , R . , Schiffmann, E . 1 98 1 . Polymorphonuclear leukocyte migration through human amnion mem brane. J. Cell. Biol. 91 :459--67 70. Bevilaqua, M. P., Pober, J. S . , Wheel er, M. E. , Cotran, R. S . , Gimbrone , M . A. 19 8 5 . Interleukin-I acts o n cultured human vascular endothelium to increase the adhesion of polymorphonuclear leu kocytes, monocytes and related leuko cyte cell lines. 1. Clin. Invest. 769:2003--20 I I 7 1 . Bevilaqua, M . P . , Pober, J . S . , Wheel er, M. E . , Cotran, R. S . , Gimbrone , M. A . 1985. Interleukin-l activation of vascular endothelium. Effects on pro coagulant activity and leUkocyte adhe sion. Am. J. Patkol. 1 2 1 :394--403 72. H arl an, J. M. 1985. Leukocyte-en dothelial interactions. Blood 65 : 5 1 3-25 73. S anchez-Madrid , F. , Nagy , J. A . , Rob bins, E . , Simon, P. , Springer, T. A . 1983. A human leukocyte differentiation antigen family with distinct alpha subunits and a COJIUDon beta-subunit: the: lymphocyte function-associated antigen (LFA-I) and C3bi complement receptor (OKM IIMac- I ) , and the p 150,95 mole cule. J. Exp. Med. 1 5 8 : 1 785-1803 74. Miller, L. J . , Schwarting, R . , Springer, T. A. 1986. Regulated expression of the Mac- I , p l50,95 glycoprotein family during leukocyte differentiation. J. Im muno!. 1 37:2891-2900 75. Ross, G. D . , Medof, M. E. 1985. Mem brane complement receptors specific for bound fragments of C3. Adv. lmmunol. 37:217-67 76. Berger, M . , O'Shea, J . , Cross, A. S . , Folks, T . M . , Chused, T . M . , et al. 1984. Human neutrophils increase ex pression of C3bi as well as C3b recep tors upon activation. J. Clin. Invest. 74: 1 566-71 77. Simon, R. H . , DeHart, P. D . , Todd, R . F. 1986. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J. Clin. Invest. 7 8 : 1 375-86 78. S impson, P. J . , Todd, R. F. III, Fan tone , J. C . , Mickelson , J. K . , Griffin, J. D . , Lucchesi, B . R. 1988. Reduction of experimental canine myocardial reperfu sion injury by a monoclonal antibody (anti-Mol , Anti-CD 1 1 b) that inhibits leukocyte adhesion. J. Clin. Invest. 8 1 :624-29 79. Jennings , R. B . , Reimer , K. A . , Steen bergen, C. Jr. 1986. Myocardial isch emia revisted. The osmolar load, mem brane damage, and reperfusion (Edito rial). J. Mol. Cell. Cardiol. 18:769-80
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MODULATION OF LEUKOCYTE-MEDIATED MYOCARDIAL REPERFUSION INJURY Benedict R. Lucchesi The University of Michigan Medical School, Department of Pharmacology, M6322 Medical Science Building I, Ann Arbor Michigan, 48109-0626 KEY WORDS:
neutrophils, free radical, ischemic myocardium, inflammatory mediators, chemotactic stimuli
INTRODUCTION New therapeutic interventions, such as coronary angioplasty or thrombolytic therapy, are directed towards the treatment of acute myocardial ischemia based upon the recognition that mortality among patients who are evolving a myocardial infarction is influenced by the extent of left ventricular dysfunc tion, which is directly related to the amount of myocardium that becomes infarcted and thus non-functional
(1).
Secondly, the size of the infarct that
results is related directly to the severity and duration of the ischemic interval. Patients who are treated and reperfused early
«4
hr) after the onset of
symptoms exhibit better functional recovery as measured by a larger ej ection fraction and smaller infarct sizes as assessed on the basis of myocardial intracellular enzyme release (2). Third, reperfusion of the acutely ischemic myocardium results in a reduction in ultimate infarct size as determined in the experimental animal and presumably in patients. Proper aggressive treatment of acute myocardial infarction should involve attempts to recannalize the obstructed coronary vessels either by mechanical means or by thrombolytic therapy
(3). 561
0066-4278/90/0315-0561$02.00
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The size of the myocardial infarct resulting from a period of regional ischemia is related directly to the severity and duration of the ischemic insult (4). In the canine heart, coronary artery occlusion for more than 4 hr followed by reperfusion results in an infarct that is essentially identical to the extent of infarction that results after permanent coronary artery occlusion for 24 hr. Coronary occlusion less than 20 min is associated with prolonged ventricular dysfunction; a phenomenon referred to as the stunned myocardium (5) that eventually recovers without evidence of myocardial cell injury. Inasmuch as the degree of myocardial cellular necrosis increases with increasing duration of ischemia, it is apparent that early reperfusion of the ischemic myocardium will result in salvage of potentially viable tissue (6). When the ischemic interval is extended to 40 min, the extent of irreversibly injured tissue (infarct size) is related directly to the duration of the ischemic event. Reimer & Jennings (4) described the "wavefront phenomenon" of advancing necrosis from the subendocardial to the subepicardial regions of the myocardium. The duration of protracted blood flow deprivation to the myocardium increased the extent of irreversibly injured myocardium and decreased the amount of tissue that could be salvaged by reperfusion. Most of the remaining viable tissue was confined to the subepicardial region, un doubtedly because of the maintenance of coronary blood flow provided by means of collateral vessels. It is clearly evident that early reperfusion of the ischemic cardiac muscle is essential if the goal is to prevent or reduce the extension of tissue damage associated with regional myocardial ischemia caused by a myocardial infarction. THE CONCEPT OF MYOCARDIAL REOXYGENATION INJURY Reperfusion of the ischemic myocardium results in an apparent acceleration of necrosis as manifested by cell swelling and the formation of contraction bands. This has been referred to as myocardial reperfusion injury. The sudden reintroduction of oxygenated whole blood reperfusion may be detrimental to the once ischemic myocyte, thereby giving rise to a paradoxical situation concerning the best approach to preserve the ischemic heart and the concept of myocardial reperfusion injury. Because the cellular morphologic changes observed with reperfusion are dependent upon the presence of molecular oxygen, it would be more appropriate to think in terms of reoxygenation injury (7). As a simple definition, myocardial reperfusion (reoxygenation) injury re fers to irreversible cellular damage (necrosis) resulting from the reintroduc tion of molecular oxygen at the time of organ reperfusion. This suggests that the reintroduction of oxygen itself causes injury that would not have occurred
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at all or at least not as rapidly as without perfusion. Injury to the myocardial cell upon whole blood reperfusion is characterized by a variety of events such as calcium overload into cells that have preexisting membrane defects, the influx of inflammatory cells (e.g. neutrophil), the generation of oxygen derived free-radicals or other long-lived oxidants and the release of proteoly tic enzymes associated with the acute inflammatory response. Reperfusion injury in vitro may be somewhat different under in vivo conditions, which involve the cumulative effects of one or more tissue or blood-borne factors. The alterations in myocardial cell viability observed under in vitro conditions may be a reflection of the intracellular generation of reactive species of oxygen and may differ significantly from what is observed in the intact heart perfused with oxygenated, whole blood. The tissue damage initiated with the reintroduction of whole blood will continue for an indeterminate period after
reperfusion has been established. The subsequent regional myocardial in flammatory response to tissue injury and the accumulation of polymorphonu clear leukocytes at the site of injury secondary to the damage inflicted upon the myocardium due to the earlier ischemic insult is responsible for the extension of the tissue damage that occurs with reperfusion (8-10). For the purpose of this presentation, we will employ a strict definition of reperfusion injury that considers the possibility that some or all of the ischemic myocytes that are not salvaged by reperfusion are viable at the time of reperfusion, but undergo a lethal explosive alteration because of an im posed oxidative stress that is associated with the reintroduction of oxygenated blood and the associated cellular components. Myocardial cells that were viable at the end of the ischemic interval are subjected to unfavorable con ditions related to reperfusion, and it is the act of reperfusion itself that leads to cell death. This is the basis for the concept of reperfusion injury as used in this review. Myocardial cell death due to reperfusion injury may be different with respect to the mechanism of death than from that which occurs from pro longed myocardial ischemia.
POTENTIAL SOURCES FOR THE PRODUCTION OF REACTIVE SPECIES OF OXYGEN
There is a complex interrelationship among the various factors that contribute to reperfusion injury. Oxygen-derived free-radicals are produced by activated neutrophils that infiltrate the ischemic and reperfused myocardium. Free radical scavengers as well as the oxygen-derived free-radical metabolizing enzyme, superoxide dismutase, and the hydrogen peroxide degrading en zyme, catalase, reduce the contribution to myocardial cell injury caused by superoxide anion and hydroxyl radical (II, 12). The polymorphonuclear
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neutrophil and other phagocytic cells possess the capacity to produce reactive species of oxygen when presented with appropriate stimuli
(13) in a metabolic
event that is referred to as the respiratory burst. The oxidants produced by the activated phagocytes consist of superoxide anion (027), hydrogen peroxide (H202), hypochlorous acid (HOCl), chloramines (RNHCl-), and hydroxyl anion (·OH). The primary function of these oxidants is to provide a defense
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mechanism against invading microorganisms. The killing of microorganisms occurs within the phagocytic vacuole in which oxidants are contained within an environment well-suited for such reactive products. The reactive species of oxygen become deleterious to surrounding cells when they are released into the extracellular environment. The primary reactant that serves as the ultimate source for each of the oxidants is 027, which results from the one-electron reduction of oxygen catalyzed by the enzyme NADPH-oxidase. The oxidase, a membrane-bound flavoprotein, which is dormant in the resting phagocytic cell, becomes activated when stimulated by components of the complement system (C5a). Neutrophils contain the enzyme myeloperoxidase, which catalyzes a H20r dependent oxidation of halide ions that involves both CI- and Br- ions to give rise to OCl- or OBr-. Each of these hypohalite anions can act as a powerful
oxidant capable of attacking a wide variety of biomolecules including
al
antiproteinase. An additional potential source of oxygen-derived free-radicals and oxidants is the enzyme, xanthine oxidase, which is localized within the vascular endothelium in many animal species studied to-date, but is not believed to be present in the myocyte of the human heart
(14). Xanthine dehydrogenase
undergoes a conversion from it s dehydrogenase to its oxidase form because of the activation of a calcium-dependent protease (14, 15). Upon reperfusion, xanthine oxidase is believed to utilize hypoxanthine as a substrate and molecular oxygen as an electron acceptor, thereby leading to the production
of superoxide anion (027). Support for this concept derives from the observa tion that allopurinol, an inhibitor of xanthine oxidase, has been reported to reduce tissue injury associated with myocardial reperfusion (15, 16). Studies that demonstrate the protective effects of allopurinol in ex perimental models of myocardial ischemia provide evidence that xanthine oxidase may participate in the generation of oxygen-derived free-radicals during myocardial reperfusion
(17). Under conditions of ischemia it appears
that the enzyme xanthine dehydrogenase is converted by a calcium-activated protease to the oxidase form, xanthine oxidase, (D to 0 conversion)
(18).
Additionally, during ischemia there is an accumulation of purine metabolites from ATP and ADP (AMP, inosine, hypoxanthine). Upon reperfusion, the required substrates hypoxanthine and the necessary electron acceptor oxygen, are provided to the enzyme, which leads to the formation of uric acid and the
REPERFUSION INJURY
565
generation of superoxide anion. The localization of xanthine oxidase, primari ly in the endothelial cell 1 ( 4), which to induce tissue injury. Consistent with this premise is the reported observation that superoxide dismutase plus catalase administered one min before reperfusion reduced both the microvascular damage and the low reflow phenomenon in the endocardium of the canine heart subjected to regional
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ischemia for
2 hr followed by reperfusion for 4 hr (19).
Kehrer et al (20) were unable to detect any significant degree of conversion of xanthine dehydrogenase to xanthine oxidase in the rat heart, and pretreat ment with allopurinol did not provide protection against the release of in tracellular enzymes when the hearts were reoxygenated after a period of hypoxia. It was concluded that the tissue injury in the hypoxic reoxygenated
rat heart was the result of a mechanism other than reactive oxygen generated et al (21),
by xanthine oxidase. A similar view was expressed by Bindoli
which indicated that the cardioprotective effect of allopurinol in the rat should be referred to as a mechanism that is unrelated to one based upon the inhibition of xanthine oxidase. Allopurinol does not prevent the formation of superoxide anion despite the fact that it can inhibit the production of uric acid by xanthine oxidase as opposed to its active metabolite oxypurinol, which can inhibit both functions of the enzyme (22). Some degree of caution is needed in interpreting data derived with allopurinol. The drug can not be regarded, when used in vivo, as a specific inhibitor of xanthine oxidase. According to more recent studies
(16), it was suggested that metabolic conversion of
allopurinol to oxypurinol may permit the active metabolite to exert an action to counteract the interaction of neutrophils with the coronary vasculature and/or with the cardiac myocytes.
THE ROLE OF REACTIVE OXYGEN METABOLITES IN REPERFUSION INJURY The first suggestive evidence of the participation of oxygen-derived free radicals in myocardial reperfusion injury in the intact animal developed from the work of Jolly et al 1 ( 1). tration of the superoxide anion metabolizing enzyme, superoxide dismutase (SOD), and the H202 degrading enzyme, catalase, to anesthetized dogs subjected to 90 min of coronary artery occlusion followed by reperfusion was effective in reducing ultimate infarct size. In contrast, the antioxidant en
40 min after reperfusion had begun. (23) subsequently showed that the administration of SOD (5 mg/kg; 3800 U/mg) alone resulted in a 50% reduction in myocardial infarct
zymes were without benefit if infused Wems et aI
size, whereas the administration of catalase alone was not accompanied with a significant degree of myocardial salvage. Ambrosio et al
(24) demonstrated
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that recombinant human superoxide dismutase could reduce ultimate infarct size in the canine heart subjected to
90 min of regional ischemia followed by
reperfusion, despite the fact that the enzyme was administered immediately upon reperfusion. The latter observation agrees with other similarly con ducted studies
(8, 11,12,25) and supports the contention that an extension of
myocardial injury occurs coincident with the onset of reperfusion. It is of
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importance to note that not all studies are in agreement. The administration of superoxide dismutase plus catalase to the canine heart subjected to either 40 (26) or 90 (27) min ischemia followed by reperfusion for 4 days failed to provide evidence for a protective effect. Several investigators who have employed the chronically instrumented canine heart also have failed to observe a reduction in ultimate infarct size with native superoxide dismutase
(28, 29) given in combination with catalase. & colleagues (28) employed a 3 hr period of ischemia followed by reperfusion for 24 hr, whereas Nejima et al (29) used a 90 min period of regional ischemia followed by 7 days of reperfusion. One biological variable that could account for the differing results among Gallagher
the various studies with SOD is the duration of occlusion used to induce the infarcts. Studies reporting positive results employed durations of occlusion of
60 or 90 min. Those reporting negative results have used ischemic intervals of 40 min or 3 hr. Reperfusion injury may require an ischemic period of greater than 40 min and less than 3 hr. The failure to substantiate a protective effect in the above mentioned studies may be model dependent. The degree of myocar dial cell death associated with a
3 hr ischemic interval may have resulted in far
too little myocardium to be salvaged as a result of the applied intervention
(30, 31). On the other hand, it is possible that an ischemic period of 40 min may be insufficient for the demonstration of myocardial injury due to a neutrophil-dependent, oxygen-radical-mediated mechanism. Eng et al
(32) provide an interesting insight into the relevance of the
duration of coronary artery occlusion in studies designed to assess in terventions for the protection of the ischemic heart. The latter investigators demonstrated that there is an explosive increase in the extent of necrosis between a narrow 50 to
60 min period of ischemia. Approximately 70% of the 90 min of severe blood flow
region at risk became necrotic when subjected to
deprivation, whereas only a limited extent of myocardial injury (approximate ly
10% of the risk region) was observed with a severe deprivation of coronary 40 min. Short periods of ischemia (e.g. 40 min) may
blood flow that lasted for
not be able to elicit a component of injury because of reperfusion, and most of the detected cellular damage is related to myocardial metabolism. Ischemia itself and the metabolic demands of the tissue at the time become the primary determinants of cell viability within the ftrst detected as a result of
20 min. Necrosis was not 20 min of severe ischemia. Beginning at 30 min of
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blood flow deprivation, necrosis progressed from the endocardium toward the epicardium in a wave front pattern so that 90 min of ischemia resulted in 70% of the risk region undergoing irreversible injury. There is support for the concept that myocardial ischemia and, in particular reperfusion injury, are accompanied by the formation of oxygen-derived free-radicals, which overwhelm diminished endogenous protective mech anisms to exacerbate cell injury. Cell death is probably a far more violent event than is often imagined, "an explosion rather than a dissolution", since the generation of free-radicals occurs rapidly, and the half-life of the reactive oxygen species, in most cases, is of relatively short duration. Recent studies utilizing electron spin resonance spectroscopy and spin trapping agents have provided direct evidence for the generation of oxygen-free radicals in myocar dial tissue during the ischemic interval and particularly during reperfusion (33-35). Short durations of ischemia are not associated with irreversible cellular changes. The detection of free-radical production became apparent during the time of coronary occlusion and increased 100-fold during reperfu sion (36). The burst of free-radical production peaked at 2� min after reperfusion and persisted for 3 hr after reflow. Relatively short periods of regional ischemia are accompanied by free-radical generation at the time of flow deprivation and by an explosive and prolonged period of free-radical production well into the period of reperfusion. The formation of both oxygen centered and carbon-centered radical species appears to be a consequence of oxy-radical attack of cardiac membrane lipids and supports the idea of lipid peroxidation in the pathogenesis of ischemic heart damage, particularly dur ing reperfusion (35). Free-radical production by endothelial cells, along with an inhibition of oxy-radical production by oxypurinol, provides additional support for the potential role of reactive oxygen products in ischemia and reperfusion injury (37). The sustained generation of cytotoxic oxygen radicals (36) may explain why interventions such as superoxide dismutase with its relatively short pharmacologic half-life of 6--10 min may appear to be ineffective in ex perimental protocols designed to assess tissue damage 4 or more days after reperfusion (26, 39, 29). The scavenging action of the enzyme would have dissipated because of its short half-life, at a time when there is a continued production of lipid peroxidation because of oxygen-free radical attack on biological membranes. Przyklenk & Kloner (41) demonstrated significant protection by superoxide dismutase in the heart subjected to 6 hr of regional ischemia without reperfusion. If perfusion was instituted, the protective effect of the scavenger was no longer apparent at 30-48 hr post reperfusion. It was concluded that superoxide dismutase delayed rather than prevented the de velopment of cell death. That this reasoning may be incorrect was suggested by a subsequent study in which, polyethylene glycol conjugated superoxide
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dismutase, with a pharmacologic half-life of ca 30 hr, not only reduced infarct size in the heart subjected to 6 hr of regional ischemia, but continued to protect against cell death when infarct size was assessed 30 hr after reperfu sion (42).
ROLE OF NEUTROPHILS AS A SOURCE OF REACTIVE OXYGEN METABOLITES AND PROTEOLYTIC ENZYMES AS POTENTIAL MEDIATORS OF MYOCARDIAL CELL INJURY Neutrophils are capable of being activated upon attachment to the vascular endothelium with the subsequent generation of toxic oxygen products and the release of destructive proteases capable of altering vascular permeability. Acid and neutral proteases are also released that can alter the integrity of the vascular wall basement membrane (43). The neutrophil contains two latent metalloproteinases, collagenase and gelatinase, that are activated by hypochlorous acid and are capable of degrading collagen in addition to lysing endothelial cells (44). Mediators present during myocardial ischemia capable of activating neutrophils include complement activation products, leukotriene B4, and platelet activating factor. It was suggested that activation of neutrophils within the vascular space results in the formation of cellular aggregates that can physically impair blood flow to the myocardial capillary bed and thereby exacerbate the ischemic injury (45). Neutrophil accumulation in the vascular bed is thought to be the reason there is sometimes inadequate reflow or the no-reflow phenomenon after brief periods of ischemia (46) followed by reperfusion. Associated with reperfusion of the ischemic myocardium is a cellular reaction characterized by the accumulation of inflammatory cells and the production of inflammatory mediators in the reperfused myocardium. The regional accumulation of inflammatory cells contributes, in part, to the extension of tissue injury associated with reperfusion of the previously ischemic myocardium (47-49). Leukocyte/endothelial cell interaction is es sential for leukocyte migration across the endothelial surface, and superoxide anion, as well as C5a, have been demonstrated to increase adhesion of neutrophils to the endothelium (50). Several studies have documented the correlation between infarct size result ing with reperfusion and the extent of neutrophil infiltration (48, 51, 52). Neutrophils have been observed to infiltrate the damaged myocardial region beginning with the onset of ischemic injury and to increase their numbers progressively for the first 24 hr post myocardial infarction (53). The infiltra tion of neutrophils within the early postischemic interval before the develop ment of myocardial necrosis has been demonstrated (54-55). There is a
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direct relationship between the duration of myocardial ischemia and the extent of neutrophil infiltration and accumulation in the reperfused region (56). The formation of tissue edema, which accompanies cellular injury, involves in teractions dependent upon the neutrophil for the full expression of the in tlammatory response (57). The generation of oxygen-derived free-radicals represents one of the main mechanisms by which polymorphonuclear neutro phils can mediate tissue injury. The reactive products of oxygen are formed during the engulfment of particulate matter or when the neutropbils are activated by soluble stimuli, particularly C5a. Romson et al (52) demonstrated that the myocardial protective effect of the non-steroidal anti-inflammatory agent, ibuprofen, was related to the inhibi tion of neutrophil accumulation in the reperfused myocardium or risk region. Evidently ibuprofen reduced myocardial infarct size in the dog subjected to 60 minutes of coronary occlusion followed by 24 hr of reperfusion. The anti inflammatory effects of ibuprofen resulted in decreased neutrophil accumula tion in the myocardial risk region witb a concomitant salvage of tissue in the area subjected to reperfusion. The protective effects of ibuprofen are un related to its ability to inhibit cyc100xygenase since neither acetylsalicylic acid nor indomethacin reduced ultimate infarct size or inhibited the release of superoxide anion by activated neutrophils (58). Ibuprofen, in contrast to indomethacin and acetylsalicylic acid, provides a protective effect via a pharmacologic mechanism that does not involve its ability to inhibit the formation of prostanoids and most likely is acting by a direct action upon the intlammatory cells by impairing their cbemoattraction to the region and impairing their ability to form and release cytotoxic derivatives. Pharmacologic interventions capable of inhibiting the lipoxygenase path way of arachidonic acid metabolism also reduce myocardial infarct size probably through a mechanism related to the inhibition of neutrophil accumulation within the myocardium. The dual cyclooxygenase-lipoxygenase inhibitor BW 755C (10 mg/kg) reduced myocardial infarct size by 50% as compared to non-treated controls when evaluated with a canine model of 90 min of coronary occlusion followed by 24 hr or reperfusion (55, 59) in association with a reduction in neutrophil accumulation in the reperfused myocardium (55),
CHEMOTACTIC STIMULI AND ACTIVATION OF NEUTROPHILS In order for neutrophils to injure ischemic tissue, there must be a chemical signal or chemoattractant to direct the neutrophil to the affected myocardial region. Ischemic myocardial tissue gives rise to a tissue protease that activates the third component of complement (60). Extensive confirmation dem-
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onstrates that myocardial ischemia activates the complement system and that ischemia results in the migration of neutrophils into the jeopardized myocar dium (61-63). Other chemotactic factors are generated in response to myocar dial tissue injury, including leukotriene B4. A chemotactic factor can be produced by a superoxide anion acting upon a plasma protein that has been characterized as a derivative of arachidonic acid (64). An increase in chemotactic activity is noted in the coronary sinus blood of dogs subjected to ligation of the left anterior descending coronary artery and correlates with the enzymatic and electrocardiographic indices of myocardial ischemia (65). Additional evidence for the participation of the complement system in the development of myocardial ischemic injury is obtained from studies in which complement depletion with cobra venom factor after coronary ligation re sulted in a reduction in the amount of myocardium that eventually became infarcted (66). These observations were confirmed with the use of im munohistochemical methods demonstrating an extensive localization of com plement components (C3, C4, and CS) within the ischemic myocardium which did not occur after decomplementation of the animal (61, 62). The membrane attack complex (MAC or attack sequence) of complement (C5b-9) is present within the ischemic myocardium of acutely infarcted humans as early as 6 hr after the onset of symptoms (67). The membrane attack complex consists of a stable macromolecular complex (C5b and C6, C7, and C8) that inserts into the membrane bilayer (with C9) and forms a pore or channel through the membrane that permits bidirectional flow of ions and macro molecules ultimately resulting in cell lysis. Thus, complement may have a direct as well as an indirect (neutrophil) effect on the sequential events associated with myocardial tissue injury and subsequent cell death. THE ROLE OF THE ENDOTHELIAL CELL
A pathophysiologic finding in myocardial ischemic injury is the presence of endothelial damage to the coronary vascular bed, which leads to the ex travasation of plasma and cellular components into the interstitial space. Polymorphonuclear leukocytes can adhere and migrate through the endothe lial cell layer and are of pathophysiologic relevance with respect to their potential role in mediating myocardial injury. This latter event is a time dependent process in the presence of total vessel occlusion. The inflammatory cells enter the damaged myocardium slowly and become apparent at 24 hr after the onset of injury. On the other hand, reperfusion of the once ischemic tissue allows for rapid access of the inflammatory cells to the jeopardized myocardial region (68). The movement of neutrophils and subsequent release of proteinases across the vascular wall may in itself be associated with permeability changes and injury to the vessel. The migration of the neutrophil
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across the connective tissue barrier of the blood vessel is dependent upon the action of proteolytic enzymes, which remain active even in the presence of plasma antiproteases (69). The adhesion of neutrophils to endothelial cells in culture is stimulated with endotoxin, interleukin-l (lL-l), or tumor necrosis factor, which suggests that the endothelial cell plays an important role in regulating the entry of the inflammatory cells to sites of tissue injury. Be vilaqua & co-workers (70) have shown that there are subtle and profound interactions between leukocytes and the vascular endothelium that are under the influence of immune modulation. It was suggested that IL-I can activate the vascular endothelium to increase the expression of its adherence mole cules for leukocytes (71). These molecules are distinct from the CDw18, LFA (Mol, gp150,95) class of glycoproteins and appear to be independent of the leukocyte receptors for the formyl peptides such as f-Met-Leu-Phe or the complement receptors for C3b or C5a. ADHERENCE PROMOTING CELL SURFACE GLYCOPROTEIN COMPLEXES A group of neutrophil cell-surface glycoproteins mediates cell-cell in teractions (72). The heterodimeric glycoprotein referred to as Mol and Mac-l antigen consists of an alpha chain, CD lIb (165 kd); and a beta subunit, CD 18 (96 kd). The members of the leukocyte family each possess the same beta subunit that is covalently linked to the alpha subunit. The latter will vary, thereby giving rise to a family of membrane glycoproteins, LFA-I, Mol and gp150,95 (73). The alpha subunits dictate the molecular specificity. The expression of these molecules on the cell surface will be determined by the origin and stage of maturation of the leukocytes (74). Activation of leukocytes by con A, FMLP, zymosan-activated serum, C5a, or calcium ionophore results in an enhanced expression of cell-surface adhesion glycoprotein mole cules in which upregulation is the result of a translocation of preformed receptors to the plasma membrane from an intracellular site. The process of expression on the cell surface is rapid and is not dependent upon new protein synthesis, which results in a 5-1O-fold increase in the number of glycoprotein receptors from the resting state of 10,000 to 20,000 molecules per cell (75, 76). Pathophysiologic conditions occur in which there is a chemoattraction and an enhanced binding of neutrophils to the surface of the affected cells. The localization of complement components may be a prerequisite for the attach ment of neutrophils to the vascular endothelium after an ischemic insult to the tissue. Mol receptors on the neutrophil bind C3bi and thus promote the adherence to cells coated with C3bi. If neutrophil adhesion to the target tissue is prevented with an anti-Mol monoclonal antibody, there is an associated
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reduction in the extent of tissue injury
(77). Neutrophil activation is accom
panied by the expression of the adhesion promoting receptors, the net result of which is an amplification of the process of neutrophil accumulation at the endothelial surface.
The resulting microenvironment at the neutrophil
endothelial cell-cell interface becomes the site at which the cytotoxic in flammatory mediators can exert their effects uninhibited by blood transported
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antioxidants and other cytoprotective factors.
THE INHIBITION OF NEUTROPHIL ADHESION 45 min after the induction of region al myocardial ischemia results in a significant protection against reperfusion injury as measured by the size of the myocardial infarct that results after 6 hr of reperfusion (78). There was no effect of the antibody on arterial blood
Administration of the anti-Mo l antibody
pressure, heart rate, or coronary artery blood flow that could account for the protective action. Anti-Mol administration in vivo significantly reduced neu trophil accumulation within the ischemic myocardial tissue as assessed by histological analysis of tissue sections. The specificity of the monoclonal antibody for the Mol receptor provides supportive evidence that the infiltrat ing neutrophil is a major contributor to the observed extension of myocardial injury at the time of reperfusion.
CONCLUSIONS It is now accepted that early reperfusion of the once ischemic myocardium is an important consideration in an effort to reduce overall loss of functional tissue within the region of myocardium at risk. Thus reperfusion is essential for survival of the jeopardized heart muscle, and the reduction in ultimate infarct size is associated with a decrease in morbidity and mortality. There will exist within the myocardial region at risk, a population of myocardial cells that are reversibly injured as a consequence of a period of (less than 3 hr) of ischemia. Within this population of cells, a fraction of the affected myocytes might undergo irreversible changes (cell death) associated with the onset of reperfusion. Reperfusion injury, therefore, refers to cell death caused by reperfusion. It is suggested that the lethal event may be reduced or prevented by appropriate measures directed against the cytotoxic effects of reactive species of oxygen and perhaps proteolytic products derived from inflammatory cells that are attracted to the injured area as a result of the influence of complement- and arachidonic-acid-derived chemoattractants, in terleukin-l, and adhesion promoting neutrophil glycoproteins. Among the sites of production of the oxygen-derived free-radicals, one might include phagocytic cells, myocardial tissue, especially the mitochondria of the reper-
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fused myocyte, and the vascular endothelial cells that possess the enzyme xanthine oxidase. Both oxygen metabolite-dependent and -independent pro cesses may participate in the explosive tissue injury that is associated with the onset of reperfusion and additive to the myocardial damage that is secondary to the ischemic insult itself. The two events may be attributed to entirely different mechanisms of cell injury. This view may be at odds with the observations reported by others (79), and additional effort must be devoted towards a better understanding of the role of the inflammatory mediators that are attracted to the myocardial risk region within moments of the onset of reperfusion. ACKANOWLEDGMENTS
The original work reported from the author's laboratory was supported by a grant from the National Institutes of Health, Heart, Lung and Blood Institute, HL-19782-1O. The author acknowledges the valuable contributions of his colleagues Dr. Paul Simpson, Dr. Steven Werns, Dr. Robert Todd III, and Dr. Joseph Fantone. Literature Cited I. Rude . R. E Muller. 1. E BraunwaJd. E. 198 I. Efforts to limit the size of myocardial infarcts. Ann. Intern. Med. 95:736-61 2. Schwarz, F., Schuler, G., Katus. H., .•
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Hofman, M., Manthey, J., ct al. 1982. Intracoronary thrombolysis in acute myocardial infarction: Duration of isch emia as a major determinant of l ate re sults after recanalization. Am. J. Car dial. 50:933-37 3. Braunwald, E. 1985. The aggressive treatment of acute myocardial infarction. Circulation 71:1087-1092 4. Reimer, K., Jennings, R. 1979. The "wave-front phenomenon" of ischemic c ell death. n. Transmural progression of necrosis within the framework of ischemic bed size (myocardium at risk) and collateral blood flow. Lab. Invest.
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McRitchie, R. J., Ma roko . P. R., Vat ncr, S. F. 1975. Regional myocardial functional and electrophysiological al terations after brief coronary artery occlusion in conscious dogs. J. Clin. Invest. 56:978-85 Koren, G., Weiss, A. T .• Hasin, Y .• Appelbaum, D., Wellber, S., et al. 1985. Prevention of myocardial damage in acute myocardial ischemia by early treatment with intravenous streptokinase. New Engl. J. Med. 313:1384--89
7. Hearse. D. J .• Hu mphrey . S. M .• Nay ler. W. G., Slade, A., Bordu, D. 1975. Ultrastructural damage associated with reoxygenation of the anoxic myocar dium. J. Mol. Cell. Cardiol. 7:315-24 8. Tamura, Y., Chi, L., Driscoll, E. M., Hoff, P. T., Freeman, B. A., et al. 1988. Superoxide dismutase conjugated to polyethylene g ly col provides sus tained pr otect ion against myocardial ischemialreperfusion injury in the canine heart. Cire. Res. 63:944--59 9. Simpson, P. J., Fantone, J. C., Luc chesi, B. R. 1988. Myocardial ischemia and reperfusion injury: Oxygen radicals and the role of the neutrophil. In Oxygen Injury, ed. B. Hal liwell. pp. 63--77. BetheSda, Md: Fed. Am. Soc. Exp. BioI. 10. Jacob, H. S., Vercellotti. G. M. 1988. Granulocyte mediated endothelial in jury: Oxidant damage amplified by lac torferrln and platelet activating factor. In Oxygen Radicals and Tissue Injury, ed. B. Halliwell, pp. 57--62. Bethesda, Md: Fed. Am. Soc. Exp. BioI. 11. Jolly, S. R., Kane, W. J., B ai lie , M. B., Abrams, G. D., Lucchesi, B. R. 1984. Canine myocardial reperfusion injury: Its reduction by the combined adminis tration of superoxide dismutase and cata lase. Res. 54:277-85 12. Werns. S. W., Simpson, P. J., Mickel son , J. K . , Shea, M. J .• Pitt, B., Luc-
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"
oxide dismutase: Insights into the pathophysiology of reperfus i on injury. Circulation 74:1424-33 25. Tamura, Y., Saito, S., Hatano, M., Lucchesi, B. R. 1989. Limitation of myocardia l infarct size by polyethylene glycol-conjugated superoxide dismutase in a canine model of 90 min coronary occlusion followed by 4 day s of reperfu sion. Kokyu To Junkan 37:201-8 26. Uraizee, A., Reimer, K. A., Murry, C. E., Jennings, R. B. 1987. Failure of superoxi de dismutase to limit myocar dial infarct size in a 40 minute ischemia! four days reperfusion model in dogs. Circulation 75: 1237-48 27. Richar d V. J., Murry, C. E., Jennings, R. B. , Reimer, K. A. 1988. Therapy to reduce free radicals during early reperfu sion does not limit the size of myocardial infarcts caused by 90 minu tes of i sch emia in dogs Circulation 78:473--80 28. Gallagher, K. P., Buda, A. J., Pace, D., Gerren, R. A. , Shlafer, M. 1986. Fail ure of superoxi de dismutase and catalase to alter size of infarction in dogs after 3 hours of oc clusio n followed by reperfu sion . Circulation 73:1065-1076 29. Nejima, J. , Canfiel d, D. R., Manders, W. T., Knight, D. R., Cohen, M. V., et al. 1989. Failure of superoxide dis mutase and catalase to alter the size of infarcts and functional recovery in con scious dogs with reperfusion. Circula tion 78:143--5 3 30. Jolly, S. R., Kane, W. J. , Hook, B. G., Abrams G. D., Kunkel, S. L., Luc chesi, B. R. 1986. Reductionofmyocar dial infarct size by neutrop hil depletion: Effect of duration of occlusion. Am. Heart J. 112:682-90 31. Reimer, K. A. , Jennings, R. B . , Cobb, F. R., Murdock, R. R., Greenfield, J. C., et al. 1985. Animal models for pro tecting the ischemic myocardium: Re sults of the NHLBI cooperative study. Cire. Res. 56:651-65 32. Eng, C. , Cho, S . , Factor, S. M., Ki rk E. S. 1987. A nonflow basis for the vulnerability of the subendocardium. J. Am. Call. Cardiol. 9:374-79 33. Bl asig, 1. E., Ebert, B., Lowe, H. 1986. Identification of free radicals trapped d uring myocardial ischemia in vitro by ESR. Stud. Biophys. 116:35-42 34. Zweier, J. L., Flaherty, 1. T., Weis feldt, M. L. 1987. Direct measurement of free radicals generated foll owi ng reperfusion o f isc hemic myocardium. Proc. Natl. Acad. Sci. USA 84:1404-1407 35. Arroyo, C. M., Kramer, J. R., Leiboff, R. H., Mergner G. W., Dickens, B. F., ,
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REPERFUSION INJURY Weglicki, W. B. 1987. Spin trapping of oxygen and carbon-centered free radi cals in ischemic canine myocardium. J. Free Radie. Bioi. Med. 3:3 1 3- 16 36. Bolli, R. 1988. Oxygen-derived free radicals and postischemic myocardial dysfunction ("Stunned Myocardium").
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J. Am. ColI. Cardiol. 1 2: 239--49 37. Zweier , J. L. , Kuppusamy, P . , Lutty,
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G. A. 1988. Measurement of endothelial c ell free radical generation : Evidence for a central mechanism of free radical in jury in post ischemic tissue. Proc. Natl. Acad. Sci. USA 85:4046-4050 Deleted in proof Richard, V. J., Murry, C. E . , Jennings , R. B . , Reimer, K. A. 1988. Therapy to reduce free radicals during early reperfu sion does not limit the size of myocardial infarcts caused by 90 minutes of isch emia in dogs. Circulation 78:473--80 Deleted in proof Przyklenk, K . , Kloner, R . A . 1987.
Effect of oxygen-derived free radical scavengers on infarct size following six hours of permanent coronary artery occlusion: S alvage or delay of myocyte
necrosis? Basic Res. Cardiol. 82: 1 46-58 42. Chi, L., Tamura, Y . , Hoff, P. T . , M acha , M . , G allagher , K. P. , et a!. 1 989. The effect of superoxide dis mutase on myocardial infarct size in the canine heart after 6 hours of regional ischemia and reperfusion. A demonstra tion of myocardial salvage. Circ. Res. 64:665-75 43. Janoff, A . , Zelig, J. D. 1 968. Vascular injury and lysis of basement membrane in vitro by neutral protease of human leukocytes . Science 16:702-4 44 . S medly , L. A. , Tonnesen, M. G . , Has lett , c . , Guthrie, L. A . , Johnston, R. B . Jr. et al. 1 986. Neutrophil-mediated in jury to endothelial cells. Enhancement
by endotoxin and essential role of neu
trophil elastase. J. Clin. Invest. 77: 1233--43 45. Jacob, H. 1 98 1 . The role of activated complement and granulocytes in shock states and myocardial infarction. J. Lab. CUn. Med. 98:645-53 46. Engler, R . , Covell, J. W. 1987. Granu
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