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Ann Thorac Surg 1999;68:1905-1912
© 1999 The Society of Thoracic Surgeons
a Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA
Address reprint requests to Dr Lucchesi, Department of Pharmacology, University of Michigan Medical School, 1301C Medical Science Research Building III, Ann Arbor, MI 48109-0632
e-mail: benluc{at}umich.edu
Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sept 21–24, 1997.
Abstract
Reperfusion of the ischemic myocardium results in irreversible tissue injury and cell necrosis, leading to decreased cardiac performance. While early reperfusion of the heart is essential in preventing further tissue damage due to ischemia, reintroduction of blood flow can expedite the death of vulnerable, but still viable, myocardial tissue, by initiating a series of events involving both intracellular and extracellular mechanisms. In the last decade, extensive efforts have focused on the role of cytotoxic reactive oxygen species, complement activation, neutrophil adhesion, and the interactions between complement and neutrophils during myocardial reperfusion injury. Without reperfusion, myocardial cell death evolves slowly over the course of hours. In contrast, reperfusion after an ischemic insult of sufficient duration initiates an inflammatory response, beginning with complement activation, followed by the recruitment and accumulation of neutrophils into the reperfused myocardium. Modulation of the inflammatory response, therefore, constitutes a potential pharmacological target to protect the heart from reperfusion injury. Recognition of the initiating factor(s) involved in myocardial reperfusion injury should aid in development of pharmacological interventions to selectively or collectively attenuate the sequence of events that mediate extension of tissue injury beyond that caused by the ischemic insult.
Prolonged ischemia, as with the evolution of an acute myocardial infarction, coronary bypass operation, or cardiac transplantation, jeopardizes cell viability and ultimately cardiac function. Myocardial ischemia of limited duration, less than 20 minutes, followed by reperfusion is accompanied by functional recovery without evidence of structural or biochemical evidence of tissue injury. Paradoxically, reperfusion of cardiac tissue, which has been subjected to an extended period of ischemia (> 45 minutes), results in a phenomenon known as myocardial reperfusion injury.
Early reperfusion of ischemic myocardium is an accepted approach for the management of patients with acute coronary syndromes. In addition, surgical interventions requiring interruption of blood flow to the heart, out of necessity, must be followed by restoration of perfusion. Numerous experimental studies have provided compelling evidence that reperfusion, although essential for tissue and/or organ survival, is not without risk due to the extension of cell damage as a result of reperfusion itself. Thus, there exists a paradox in that tissue viability can be maintained only if reperfusion is instituted within a reasonable time period, but only at the risk of extending the injury beyond that due to the ischemic insult itself. The suggestion of this paradoxical situation was demonstrated by the observation that ventricular fibrillation was prominent when the regionally ischemic canine heart was subject to reperfusion. Subsequently, adverse structural and electrophysiologic changes associated with reperfusion of the ischemic canine heart were reported by Jennings and coworkers [1]. Later, Hearse and coworkers [2] introduced the concept of an oxygen paradox, when they noted cardiac muscle enzyme release and alterations in ultrastructure when isolated hearts were reoxygenated after a period of hypoxic perfusion.
Myocardial reperfusion injury is defined as the death of myocytes, alive at the time of reperfusion, as a direct result of one or more events initiated by reperfusion. Therefore, myocardial cell damage results from the restoration of blood flow to the previously ischemic heart thereby extending the region of irreversible injury beyond that due to the ischemic insult alone. Reperfusion injury may also be defined as "those metabolic, functional, and structural consequences of restoring coronary arterial flow that can be avoided or reversed by modification of the conditions of reperfusion [3]." The cellular damage that results from reperfusion can be reversible or irreversible, depending on the length of the ischemic insult. If reperfusion is initiated within 20 minutes after the onset of ischemia, the resulting myocardial injury is reversible and is characterized, functionally, by depressed myocardial contractility, which eventually recovers completely. Myocardial tissue necrosis is not detectable in the previously ischemic region although functional impairment of contractility may persist for a variable period, a phenomenon known as myocardial stunning. Initiating reperfusion after a duration of ischemia longer than 20 minutes, however, results in irreversible myocardial injury or cellular necrosis. The extent of tissue necrosis that develops during reperfusion is related directly to the duration of the ischemic event. Tissue necrosis originates in the subendocardial regions of the ischemic myocardium and extends to the subepicardial regions of the area at risk, often referred to as the wave-front phenomenon. The cell death that occurs during reperfusion can be characterized microscopically by explosive swelling, which includes disruption of the tissue lattice, contraction bands, mitochondrial swelling, and calcium phosphate deposits within mitochondria.
The almost instantaneous morphologic changes in cellular structure that appear upon reperfusion of the previously ischemic myocardium, are in stark contrast to the histologic picture of heart muscle that has evolved to the stage of necrosis due to an uninterrupted ischemic insult. The latter evolves over the course of hours after the onset of ischemia, whereas reperfusion injury occurs within minutes of reperfusion and is additive to that component of cell death due to the ischemic event itself. Early morphologic observations that reperfusion of severely ischemic myocardium caused structural derangements exceeding the amount of damage seen with ischemia alone, suggest that reperfusion injury is a true pathologic phenomenon. This concept has been challenged by those who express the view that reperfusion of ischemic myocardium simply accelerates the morphologic expression of irreversible injury [4, 5].
Despite the many opposing views, there are many studies in which infarct size reduction was observed when pharmacologic interventions were administered at the moment of reperfusion. On the basis of these studies, a number of potential mechanisms of reperfusion injury have been explored as possible sites for pharmacotherapy for the reduction of injury in tissues subjected to ischemia and reperfusion.
The oxygen paradox
The oxygen paradox hypothesis is based on the premise that oxygen, although essential for tissue survival, can be injurious during reperfusion of the previously ischemic myocardium. Upon reperfusion, molecular oxygen undergoes sequential reduction to form reactive species of oxygen, including superoxide anion and hydroxyl radical, in addition to hydrogen peroxide. The interaction of oxygen-derived free radicals with cell membrane lipids and essential proteins contribute to myocardial cell damage, leading to depressed cardiac function and irreversible tissue injury.
A related hypothesis is the calcium paradox which suggests that myocardial ischemia, followed by reperfusion, causes the heart to lose the ability to maintain intracellular concentrations of calcium ion within the physiologic limit. Increased intracellular calcium concentrations may occur as extracellular calcium ions accumulate by gaining access to the cell through leaky myocardial cells, possibly the result of a separation of the glycocalyx from the sarcolemmal membrane. Myocardial calcium overload can also result from dysfunctional sarcoplasmic reticulum unable to adequately sequester and store intracellular ionized calcium. The possibility exists that the generation of oxygen-derived free radicals is a direct cause of myocardial calcium overload by damaging the sarcoplasmic reticulum.
Oxygen-derived free radicals
A free radical may be defined as any atom or molecule that can exist independently with one or more unpaired electrons in its outer orbital. Because of the existence of an unpaired electron, the atom or molecule is relatively unstable and, in general, is highly reactive. In the case of oxygen, the reduction of molecular oxygen (O2) results in superoxide anion (O2-). Formation of superoxide anion is the first of several steps in forming other oxygen-derived reactive products, which include hydrogen peroxide and hydroxyl radical (·OH). In the Haber-Weiss reaction, O2 and two hydroxyl radicals are formed when O2- reacts spontaneously with hydrogen peroxide (H2O2). In the Fenton reaction (also known as the iron-catalyzed Haber-Weiss reaction), ·OH is formed by the reaction of iron (II) H2O2. Focus has turned to the generation of the free radical, peroxynitrite (ONOO-), as another possible contributor to reperfusion injury. Formation of ONOO- occurs when O2- interacts with nitric oxide (NO), the endothelium-derived relaxing factor. The reaction can continue when ONOO- becomes protonated to form peroxynitrous acid (ONOOH). The spontaneous breakdown of peroxynitrous acid (ONOOH) can provide another source of ·OH and nitrogen dioxide (NO2). Peroxynitrite is formed during reperfusion of the rat isolated heart, and may contribute to post ischemic myocardial dysfunction. When hearts were treated with the nitric oxide synthase inhibitors, L-NAME (N
-nitro-L-arginine methyl ester) or L-NMMA (LG-methyl-L-arginine), or superoxide dismutase, peroxynitrite synthesis was attenuated during the first 15 minutes of reperfusion. Likewise, the hearts were functionally protected after treatment with the NO synthase inhibitors or superoxide dismutase (SOD), suggesting that peroxynitrite formation contributes to myocardial reperfusion injury.
Several other mechanisms may contribute to oxygen-derived free radical production during myocardial ischemia and reperfusion. A number of enzymatic mechanisms provide an intracellular source of free radical generation. Among the most thoroughly studied are, xanthine oxidase, the mitochondrial cytochrome oxidase pathway, cyclooxygenase, lipoxygenase, and oxidation of catecholamines. In addition, reperfusion of the ischemic tissue leads to the rapid accumulation of neutrophils at the site of injury. Neutrophils adhere to the vascular endothelium, transmigrate to the extravascular space, and can adversely affect viable tissue through the release of proteolytic enzymes and generation of reactive oxygen species. The oxygen-derived cytotoxic products contributed by the resident neutrophils include superoxide anion and hypochlorous acid.
Regardless of the source of oxygen-derived free radicals, cellular injury can occur during reperfusion if the level of oxidative stress exceeds the capacity of endogenous free radical scavenging mechanisms. Either the endogenous antioxidants are overwhelmed, or they too, are adversely affected by the ischemic insult, thus rendering the cell subject to the damaging effects of reactive oxygen species. Based upon data from experimental studies in animals, the exogenous administration of free radical scavengers has proved beneficial in limiting the extent of tissue injury and preserving cardiac function after myocardial ischemia and reperfusion. In vivo experimental evidence suggesting that oxygen-derived free radicals had a role in myocardial reperfusion injury came from the study by Jolly and associates [6], conducted in a canine model of ischemia and reperfusion injury. In this model, the left circumflex coronary artery was occluded for 90 minutes, followed by 24 hours of reperfusion. Concomitant administration of the O2- scavenger, SOD, and the H2O2 degrading enzyme, catalase, before induction of ischemia or 15 minutes before reperfusion, resulted in a reduction of myocardial infarct size. If the combination therapy of SOD and catalase was administered 40 minutes after the initiation of reperfusion, a protective effect was not obtained. These findings favor the conclusion that free radical mediated damage occurs at the time of reperfusion. Free radical formation immediately upon reperfusion has been shown in experimental animals [7], as well as in humans with acute myocardial infarction undergoing thrombolysis [8], routine coronary angioplasty [9] or open heart operation [10].
The ability of reactive oxygen species to alter myocardial function has been demonstrated in vitro as well as in vivo. The exposure of the heart to free radical generating solutions or the addition of H2O2 to the perfusion medium, has invariably been accompanied by alterations in cardiac function and biochemical changes similar to those observed in the stunned myocardium as a result of ischemia and reperfusion. Altered myocardial function and tissue injury were noted in response to singlet oxygen generated from photosensitized rose Bengal. Generation of singlet oxygen produced a transient positive inotropic response followed by after-contractions and muscle contracture. Electrophysiologic alterations described as action potential prolongation, early after depolarizations and oscillations in the resting membrane potential, provide further evidence of altered membrane function.
The positive results from experimental studies conducted in vivo are supported by studies in which isolated hearts perfused with a crystalloid perfusion medium containing free radical scavengers were protected against injury from global ischemia and reperfusion. The low molecular weight, manganese-based SOD mimetic, SC-52608, preserved left ventricular function in a Langendorff-perfused rabbit isolated heart model of myocardial reperfusion injury. These findings have been corroborated through the use of magnetic resonance spin trapping techniques, providing direct evidence for the role of oxygen-derived free radicals as a mediator of reperfusion injury.
Not all studies with free radical scavengers have provided agreement with respect to a reduction in infarct size, or support the concept of myocardial reperfusion injury. Uraizee and coworkers [11], and Gallagher and coworkers [12], were unable to demonstrate a reduction in myocardial infarct size with SOD and catalase in the canine heart subjected to regional myocardial ischemia followed by reperfusion. Reasons for the divergent outcomes in the canine heart are not readily apparent, but have been attributed to undocumented differences in collateral blood flow between control and treatment groups, imprecise determinations of infarct size (tetrazolium method versus histologic determination), and the wide variation of collateral blood flow in the canine heart. In addition, attention must be given to the different durations of the ischemic event (40 minutes versus 90 minutes versus 3 hours) among the reported studies. Relatively brief ischemic periods (40 minutes) may not be associated with a significant component of reperfusion injury, whereas an ischemic interval of 3 hours may have induced near complete injury within the jeopardized region, due to the ischemic insult, thus leaving little opportunity to observe that component of injury associated with reperfusion.
The role of the immune system: the complement cascade
The complement system is a major component of the humoral immune system and is comprised of more than thirty proteins found in the plasma or associated with cell membranes. This system functions to initiate inflammation, destroy pathogens, aid in the clearance of immune complexes, and disrupt cell membranes.
Complement activation can occur through one of two pathways, the classical pathway and the alternative pathway, which merge at the enzymatic conversion of the complement protein, C3, to C3a and C3b, as illustrated in Figure 1. Activation of the two pathways is initiated through different mechanisms. The classical pathway is activated when the first complement protein, C1, binds to an antibody-antigen complex, whereas the alternative pathway is continuously in a low state of activation in the fluid phase. This low level of activation, known as C3 tick-over, generates indiscriminate C3b deposition on biological cells (host or foreign), acting as a positive feedback amplification loop which can act explosively in response to a variety of activators. The latter consist of certain particulate polysaccharides, fungi, bacteria, and viruses, in addition to certain mammalian cells and aggregates of immunoglobulins. Host cells are protected from further C3 tick-over, and the alternative pathway is controlled due to regulatory proteins found on host cell surfaces and in the plasma. Activation of the complement cascade through a series of enzymatic reactions results in the formation of the anaphylatoxins, C3a, C4a, and C5a, as well as the terminal complement complex or the membrane attack complex (MAC, C5b-9). The anaphylatoxins possess vasoactive properties, enhance vascular permeability and are chemotactic, thus aiding in the recruitment of polymorphonuclear leukocytes (PMNs), to a region of tissue injury. The MAC is the terminal lytic moiety which inserts into target cell membranes, mediating an influx of water and calcium ions into the cell with the potential to bring about cell lysis. Nucleated cells are capable of defense against the injury by the MAC. The latter has been attributed to an increase in cellular metabolic activity leading to inactivation of complement channels by shedding and internalization. The irreversible phase of MAC-mediated nucleated cell damage may be due to activation of calcium-dependent, membrane bound phospholipases and subsequent lethal disruption of cellular membranes.
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Hill and Ward first implicated complement as a major contributor to inflammation during myocardial reperfusion injury with their observation of a C3-split chemotactic fragment generated in infarcted rat myocardium [13]. Since then, other studies have investigated the role of complement in myocardial ischemia and reperfusion injury. An interesting observation is that ischemia-induced complement activation, as evidenced by the deposition of complement components, is not apparent immediately and requires approximately 45 minutes. This would suggest that ischemic insults of limited duration, in contrast to longer intervals of ischemia, may not induce injury associated with complement activation. In the absence of reperfusion, MAC accumulates in the ischemic cardiac muscle after several hours of coronary artery occlusion, whereas 30 minutes of ischemia followed by reperfusion results in significant increases in MAC deposition within 15 minutes of reperfusion. Localization of complement proteins in most infarcted myocardial fibers and vessels coincided with the accumulation of polymorphonuclear leukocytes. Neither deposition of complement proteins nor leukocytes, however, is observed in myocardial tissue that is not subjected to an ischemic insult.
Infarct size was significantly less in rabbits genetically deficient in the complement protein, C6 compared to C6-competent rabbits. Likewise, the no-reflow phenomenon and arrhythmias associated with ischemia and reperfusion were significantly less in the C6-deficient rabbits, suggesting that MAC has an important role in early reperfusion injury. Antibodies directed against the neoantigen of the human C5b-9 complex have been used to identify MAC deposition in infarcted human myocardial tissue obtained at autopsy. Little detection of the MAC was observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. The data suggest that the initial ischemic insult may cause alterations in the ability of the cardiac myocytes to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes could contribute to functional disturbances (signal transduction) and irreversible damage (altered intracellular electrolyte and water balance) of myocardial cells during ischemia and reperfusion. Other studies have shown a significant increase in the deposition of C5b-9, C3bBb, and the degradation products of the anaphylatoxins in the plasma of patients after acute myocardial infarction. The findings support the concept that complement activation is involved during myocardial ischemia and the evolution of an acute myocardial infarction [14, 15].
The neutrophil and endothelium
The neutrophil represents a major cellular component of the inflammatory response contributing to myocardial reperfusion injury. The role of the neutrophil in myocardial ischemia and reperfusion injury was first demonstrated by histologic studies showing a direct correlation between the duration of ischemia and infarct size with the extent of neutrophil accumulation within the myocardial tissue [16]. Induction of neutropenia, as well as the inhibition of neutrophil adhesion [17], result in a cardioprotective effect, further implicating the importance of neutrophils in contributing to the development of myocardial reperfusion injury. Tissue injury results in a localized inflammatory response as manifest by the accumulation of polymorphonuclear leukocytes. The neutrophil is directed to the site of inflammation by sequential formation of chemotactic factors which include the complement fragment C5a, interleukin-8 (IL-8), and transforming growth factor-
(TGF-). Neutrophil chemotaxis occurs over a concentration gradient, where the chemotactic factor concentration is highest at the site of inflammation and can serve as a neutrophil activator at higher, localized concentrations. The source for the chemotactic agents are the neutrophils themselves, acting in a paracrine-like feedback manner. Leukocyte chemotactic factors also can be produced by activated endothelium and myocardial tissue. The second component necessary for neutrophils to participate in inflammation involves the endothelium. The endothelium serves two purposes: (1) the chemotactic concentration gradient is established along the extracellular matrix on the surface of the vascular endothelial cells; and (2) the endothelium serves as a matrix by which the neutrophil migrates to a site of inflammation through a series of orchestrated events involving adhesion molecules.
The ability of chemotactic agents to bind to the endothelial extracellular matrix (composed of proteoglycans) is an important prerequisite for the establishment of a chemotactic gradient and activation of the neutrophil. The extracellular matrix of the endothelium functions to sequester the chemotactic factor, thereby concentrating the agent along the endothelium. The extracellular matrix of the endothelium also serves to present chemotactic factors in the proper configuration for subsequent neutrophil activation. This is exemplified by studies where neutrophils activated by IL-8 before contacting the endothelium lost their ability to adhere to the vessel walls and became inactivated through premature stimulation. The endothelium serves as an efficient system to capture and present leukocyte chemotactic stimuli to ensure their proper activation during initiation of an inflammatory response to injury. Without the endothelium, the neutrophil can not properly position itself for activation during the inflammatory process.
Endothelial and neutrophil-derived adhesion molecules serve important roles in properly orienting the neutrophils, temporally and spatially, for activation along the endothelium. Leukocyte activation is a multistep process which is observed best in the postcapillary venule where the inflammatory cells leave the circulation and migrate toward the site of tissue injury. Initial rolling, followed by spreading and firm adhesion, and diapedesis are the steps required for neutrophil recruitment. Interference with of any one of these steps can serve as potential therapeutic targets in reducing neutrophil-mediated tissue injury through modulation of the inflammatory response.
The first step in neutrophil recruitment involves rolling along the endothelium. This phase of neutrophil recruitment serves to slow neutrophils from the general circulation. The selectin adhesion molecules are responsible for the loose interaction between the endothelium and neutrophils, tethering the neutrophil to the endothelial cell surface. Three adhesion molecules belong to this family of selectins: E-selectin (ELAM-1), L-selectin (LAM-1, LECAM-1, Mel-14) and P-selectin (GMP-140, PADGEM). L-selectin is expressed constitutively on the surface of neutrophils and monocytes, whereas E-selectin and P-selectin are mainly expressed on activated endothelial cells. P-selectin is also found on activated platelets. The expression of E-selectin and P-selectin is inducible. E-selectin is synthesized transiently and transported to the endothelial cell surface in response to endothelial cell activation by an inflammatory mediator. P-selectin is constitutively synthesized and stored in the Weibel-Palade bodies of the endothelial cells from which it is mobilized rapidly to the endothelial surface in response to an appropriate inflammatory mediator.
L-selectin, on the surface of the neutrophil, serves a role in neutrophil rolling along the endothelium. The counter-ligands for the selectins are not definitively known, but it is believed that the counter-receptors or ligands for P-selectin and L-selectin are the cell surface carbohydrate moieties, sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA). P-selectin and L-selectin appear to function early in the course of neutrophil-mediated reperfusion injury, whereas the participation of E-selectin occurs at a later time period. Several studies using blocking antibodies against the P-selectins and L-selectins have demonstrated a protective effect against reperfusion injury in several animal models of experimental ischemia and reperfusion injury.
The second step in neutrophil recruitment and activation is firm adhesion, where the neutrophil attaches to the endothelial surface after activation by an endothelial-associated activator such as platelet activating factor (PAF) and/or the chemotactic factors, C5a and IL-8. Firm adhesion of the neutrophil includes the shedding of L-selectin adhesion molecules and the mobilization of CD11/CD18 ß2 integrin adhesion molecules from intracellular stores. Platelet activating factor (PAF), in conjunction with P-selectin, acts to upregulate the neutrophil’s CD11/CD18 complex, which initiates the transition state of the neutrophil from rolling to firm adhesion. The CD11/CD18 complex is a heterodimeric glycoprotein comprised of an and ß subunit. These glycoproteins possess a common ß subunit (CD18) noncovalently bound to one of three distinct subunits: CD11a (LFA-1), CD11b (Mo1, Mac-1) or CD11c (gp150). The CD11/CD18 (ß2) integrin adhesion molecule on the neutrophil binds to the endothelium-associated complement opsonizing particle, iC3b mediating a firm adhesion between the neutrophil and endothelial cell. The opsonizing particle, iC3b, however, is not the only counter ligand for CD11/CD18. Endothelium-derived adhesion molecules known as intercellular adhesion molecules (ICAMs) (part of the IgG superfamily), also act as counter ligands for the CD11/CD18 complex to mediate firm adhesion on the endothelium. The iC3b complement derived protein mediates firm neutrophil adhesion acutely during reperfusion whereas ICAMs are responsible for further recruitment of neutrophils in the later phases of the inflammatory response. Limitation of infarct size, in studies utilizing antibodies directed against the CD11b alpha subunit, have demonstrated the importance of the CD11b moiety in neutrophil-mediated reperfusion injury [17].
Once firm adhesion has occurred, the neutrophil can proceed through the last stages of recruitment, which is diapedesis or transendothelial migration through intercellular junctions of adjacent endothelial cells. Transendothelial migration is dependent upon interactions between neutrophil CD18 and integrins in the endothelial intercellular junctions. Although several studies have demonstrated the importance of CD18 in diapedesis, it is unclear as to which integrins are important in the process of diapedesis [18]. The neutrophil gains further access into the extravascular compartments by releasing proteases (ie, collagenase and elastase), oxygen metabolites (O2-, ·OH, H2O2), hypochlorous acid and other cytotoxic substances, thereby further increasing tissue injury.
Interactions between complement and the neutrophil
The complement system and the neutrophils are distinct arbiters of the inflammatory process, each contributing to reperfusion injury. The complement system, however, has direct and indirect influence on neutrophil activation and recruitment during myocardial infarction. Recognizing that C5a facilitates the adhesion of polymorphonuclear leukocytes to the vascular endothelium in the inflammatory zone, Simpson and colleagues [19] inhibited the C5a-induced recruitment of neutrophils to the ischemic myocardium in a canine model of regional ischemia using an analogue of prostacyclin. Furthermore, a monoclonal antibody to C5a, that inhibits neutrophil cytotoxic activity, but neither affects formation of the membrane attack complex nor myocardial neutrophil accumulation, decreased infarct size in pigs [20]. The reported observations support the concept of an important role for the alternative complement pathway and C5a in the propagation of cardiac damage during reperfusion. Other investigators [21] demonstrated the presence of neutrophil chemotactic activity in cardiac lymph fluid collected during reperfusion in the canine heart subjected to a period of regional myocardial ischemia and infarction. The ability of postischemic cardiac lymph to alter neutrophil function was prevented by rabbit anti-canine C5a.
The anaphylatoxins, C3a and C5a, generated subsequent to complement activation during reperfusion, have profound effects on the coronary vasculature and serve as potent chemoattractants and activators for cellular constituents of inflammation (mainly neutrophils). In the rat isolated heart, in which the effects of isolated cellular or humoral factors could be examined, significant alterations in left ventricular developed pressure (LVDP) occur with no measurable free radical generation. Reperfusion with plasma or neutrophils alone did not alter postischemic LVDP, whereas plasma and neutrophils together caused marked injury. Electron paramagnetic resonance measurements with the spin trap 5,5-dimethyl-pirroline-N-oxide (DMPO) in the absence of neutrophils demonstrated that oxygen free radical generation occurred only during the first 1 to 2 minutes of reflow. Upon reperfusion with neutrophils and plasma, however, radical generation persisted for more than 10 minutes. Increased neutrophil accumulation was observed in the postischemic heart in the absence of plasma, however, plasma factors were required for neutrophil-mediated contractile failure. C5a alone did not cause significant injury, but in the presence of neutrophils it effectively substituted for plasma, causing marked injury. Thus, plasma factors, most likely complement, are required for neutrophil activation with oxygen free radical generation and secondary contractile dysfunction.
In addition to direct activation of neutrophils through C5a generation during the reperfusion period, complement activation indirectly increases neutrophil recruitment and infiltration into the myocardial risk region. Formation and deposition of the terminal complex of complement activation in ischemic myocardial tissue has been verified by several studies. Mathey and coworkers [22] investigated the time course of C5b-9 deposition and the influence of reperfusion. In the absence of reperfusion, C5b-9 accumulation in the ischemic myocardium was found after only 5 to 6 hours of coronary artery occlusion. In the group with ischemia and reperfusion, significant C5b-9 deposition was already observed after 30 minutes of myocardial ischemia. In the absence of reperfusion, C5b-9 accumulation occurs as a late event when most of the jeopardized myocardium has probably already become irreversibly injured. In the presence of reperfusion the complement system is activated rapidly, and could have an important role in the pathogenesis of reperfusion injury.
The experimental data support the conclusion, that myocardial injury, in response to ischemia and reperfusion, is associated with a sequence of events in which the complement system has a central role. The assembly on the target cell of the membrane attack complex, C5b-8 and polymeric C9, traverses the cell membrane thereby allowing osmotic leakage from the cell and its ultimate destruction. In addition to the direct lytic attack on a susceptible target, the complement system mediates the inflammatory response beginning with the opsonization of the target tissue through deposition of iC3b on the cell membrane, which together with the anaphylatoxin, C5a-induced activation of the neutrophils, directs the inflammatory cells to the site of injury and their emigration across the vascular bed into the interstitial space. Inflammation is an ordered process, mediated by the sequential upregulation of intercellular adhesion receptors and their complimentary ligands on the endothelial cells and leukocytes, with the neutrophils being the first cells to appear at the acute inflammatory site. In considering the role of the complement system, it is essential to recognize the temporal relationship between the ischemic event and the activation of the complement cascade. Relatively brief periods of myocardial ischemia, although capable of inducing varying degrees of irreversible ischemic cell injury, may not be of sufficient duration to achieve activation of the complement cascade. Therefore, the tissue injury associated with reperfusion may not manifest in the absence of assembly of the membrane attack complex, or the orchestration of an inflammatory response mediated by opsonization of the target and activation of the neutrophils. The explosive manner in which reperfusion injury is manifest is in contrast to the slow and progressive cell death associated with persistent ischemia. These observations are in keeping with the rapid appearance of the MAC upon reperfusion, as opposed to little to no appearance of the MAC with continuous ischemia until many hours have elapsed. Thus, modulation of complement activation should represent a potential approach to limiting neutrophil-dependent tissue damage during myocardial ischemia and reperfusion injury.
Glycosaminoglycans and reperfusion injury-modulation of the complement cascade
Heparin, a glycosaminoglycan, consists of several linear polysaccharide chains and is a major component of proteoglycans found in abundance especially on the glycocalyx lining the vascular lumen [23]. This highly sulphated, negatively charged molecule consists of repeating units of glucosamine and hexuronic acid (ie, iduronic and glucuronic acid) and is produced endogenously by mast cells and basophils. Commercial heparin is extracted from porcine intestinal mucosa or bovine lung (molecular mass from 4 to 30 kDa), and known best for its ability to augment antithrombin III activity, thereby inhibiting coagulation. Because of its ability to inhibit coagulation, heparin has been used clinically in the prevention of postoperative thrombosis and the treatment of acute venous thrombosis since its introduction more than 50 years ago. Black and colleagues [24] evaluated the effects of heparin and a non-anticoagulant analog of heparin, N-acetylheparin, in a canine model of ischemia and reperfusion injury. Heparin and N-acetylheparin both reduced infarct size after 90 minutes ischemia followed by 6 hours of reperfusion when administered 75 minutes after occlusion of the left circumflex coronary artery and again at 1.5 and 3 hours after reperfusion. The aPTT, or bleeding times, in dogs treated with N-acetylheparin did not change compared to controls, but as expected, were increased in animals receiving heparin. Thus, the cardioprotective effect mediated by the glycosaminoglycans was independent of any anticoagulant action and was postulated to derive from inhibition of complement activation.
Other studies have demonstrated that glycosaminoglycans do not have to be administered immediately before reperfusion or continuously during reperfusion in order to observe a cardioprotective effect from ischemia/reperfusion injury. When heparin or N-acetylheparin was administered 2 hours before induction of regional ischemia, followed by 6 hours of reperfusion, infarct size was reduced by approximately 33% and 43%, respectively, when compared to controls. Pretreatment with either heparin or N-acetylheparin prevented myocardial dysfunction in the rabbit isolated heart subjected to global ischemic arrest and reoxygenation [25]. In the latter case, treatment was administered to the intact rabbit, and the ischemia and reperfusion applied to the isolated heart 2 hours after treatment of the heart donor, suggesting that the glycosaminoglycans provide a protective effect by binding to the myocardium.
Heparin and related glycosaminoglycans are known to inhibit the complement cascade at multiple sites in both the classical and alternative pathways [23]. In addition, heparin is reported to inhibit neutrophil adhesion to endothelial cells, in vitro, and reduce neutrophil superoxide generation, phagocytosis, and chemotaxis. It may be one or a combination of these properties of glycosaminoglycans that account for reducing the extent of myocardial damage associated with ischemia and reperfusion.
Myocardial ischemia and reperfusion injury is accompanied by an inflammatory response contributing to reversible and irreversible changes in tissue viability and organ function. Endothelial and leukocyte responses are involved in tissue injury, orchestrated primarily by the complement cascade. Anaphylatoxins and assembly of the MAC contribute directly and indirectly to further tissue damage. Tissue salvage can be achieved by depletion of complement components, thus making evident a contributory role for the complement cascade in ischemia and reperfusion injury. The complexity of the complement cascade provides numerous sites as potential targets for therapeutic interventions designed to modulate the complement response to injury. The latter is exemplified by the ability of a soluble form of complement receptor 1 (sCR1) to decrease infarct size in in vivo models of ischemia and reperfusion injury as well as prevent myocyte and vascular injury and organ dysfunction by interdicting assembly of the MAC. Effective inhibitors of the complement are not limited to newly developed compounds or solubilized forms of endogenous regulators of complement activation. Therapeutic agents in common use, such as heparin and related nonanticoagulant glycosaminoglycans, are known to inhibit the complement activation in vitro, as well as in vivo, and may prove useful as cytoprotective agents. The discussion has emphasized the need to employ experimental models representative of clinical settings in which complement activation is associated with tissue injury from the standpoint of organ function and cell viability. Furthermore, there is the need to identify pharmacologic interventions capable of inhibition of the human complement system before recommending such agents to human clinical investigation. Towards this end, we have demonstrated the use of a relatively simple experimental model that makes use of human plasma (source of human complement) and a discordant tissue serving as the target for complement mediated cytotoxic events. The data obtained with a specific pharmacologic intervention, are then correlated using in vivo animal models of myocardial ischemia and reperfusion injury. There has been a renewed emphasis on the complement system in recent years, resulting in new pharmacologic interventions that are in the early stages of development, and await the opportunity to enter clinical trial. Increased knowledge of disease states associated with inappropriate activation of the complement system is being sought, with the hope that a pharmacologic approach to therapy becomes a reality.
While the long-term effects of complement inhibition remain to be determined, the results summarized from the aforementioned studies provide a convincing argument for limited duration inhibition of the complement cascade as a therapeutic tool for the reduction of cellular injury arising from a diverse array of pathophysiologic conditions. Among the areas of consideration is that which involves the inflammatory response to tissue and organ ischemia that is exacerbated further by reperfusion. The development of pharmacologic interventions used to modulate the complement cascade, represents an area that is of import and in need of continued research.
References
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P. Rocic, C. Kolz, R. Reed, B. Potter, and W. M. Chilian Optimal reactive oxygen species concentration and p38 MAP kinase are required for coronary collateral growth Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2729 - H2736. [Abstract] [Full Text] [PDF]
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J. L. Zweier and M.A. H. Talukder The role of oxidants and free radicals in reperfusion injury Cardiovasc Res, May 1, 2006; 70(2): 181 - 190. [Abstract] [Full Text] [PDF]
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J. P. French, J. C. Quindry, D. J. Falk, J. L. Staib, Y. Lee, K. K. W. Wang, and S. K. Powers Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H128 - H136. [Abstract] [Full Text] [PDF]
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Z. Xu, S.-S. Park, R. A. Mueller, R. C. Bagnell, C. Patterson, and P. G. Boysen Adenosine produces nitric oxide and prevents mitochondrial oxidant damage in rat cardiomyocytes Cardiovasc Res, March 1, 2005; 65(4): 803 - 812. [Abstract] [Full Text] [PDF]
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D. A. Lauver, E. A. Booth, A. J. White, E. Poradosu, and B. R. Lucchesi Sulodexide Attenuates Myocardial Ischemia/Reperfusion Injury and the Deposition of C-Reactive Protein in Areas of Infarction without Affecting Hemostasis J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 794 - 800. [Abstract] [Full Text] [PDF]
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M. Marchesi, E. A. Booth, T. Davis, C. L. Bisgaier, and B. R. Lucchesi Apolipoprotein A-IMilano and 1-Palmitoyl-2-oleoyl Phosphatidylcholine Complex (ETC-216) Protects the in Vivo Rabbit Heart from Regional Ischemia-Reperfusion Injury J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 1023 - 1031. [Abstract] [Full Text] [PDF]
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T. A. Khan, C. Bianchi, P. Voisine, J. Feng, J. Baker, M. Hart, M. Takahashi, G. Stahl, and F. W. Sellke Reduction of myocardial reperfusion injury by aprotinin after regional ischemia and cardioplegic arrest J. Thorac. Cardiovasc. Surg., October 1, 2004; 128(4): 602 - 608. [Abstract] [Full Text] [PDF]
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D. A. Liem, C. C. Gho, B. C. Gho, S. Kazim, O. C. Manintveld, P. D. Verdouw, and D. J. Duncker The Tyrosine Phosphatase Inhibitor Bis(Maltolato)Oxovanadium Attenuates Myocardial Reperfusion Injury by Opening ATP-Sensitive Potassium Channels J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1256 - 1262. [Abstract] [Full Text] [PDF]
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L. B Becker New concepts in reactive oxygen species and cardiovascular reperfusion physiology Cardiovasc Res, February 15, 2004; 61(3): 461 - 470. [Abstract] [Full Text] [PDF]
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T. Ege, S. Eskiocak, V. Yuksel, A. Taskiran, and E. Duran Importance of internal mammary artery perfusion in cardiac ischemia and reperfusion Perfusion, December 1, 2003; 18(6): 351 - 356. [Abstract] [PDF]
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Z. S. Jonjev, D. W. Schwertz, J. M. Beck, J. D. Ross, and W. R. Law Subcellular distribution of protein kinase C isozymes during cardioplegic arrest J. Thorac. Cardiovasc. Surg., December 1, 2003; 126(6): 1880 - 1885. [Abstract] [Full Text] [PDF]
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M. H. Kown, M. A. Lijkwan, C. L. Jahncke, S. Murata, J. B. Rothbard, and R. C. Robbins L-arginine polymers enhance coronary flow and reduce oxidative stress following cardiac transplantation in rats J. Thorac. Cardiovasc. Surg., October 1, 2003; 126(4): 1065 - 1070. [Abstract] [Full Text] [PDF]
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F. Vetterlein, C. Schrader, R. Volkmann, M. Neckel, M. Ochs, G. Schmidt, and G. Hellige Extent of damage in ischemic, nonreperfused, and reperfused myocardium of anesthetized rats Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H755 - H765. [Abstract] [Full Text] [PDF]
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A. J. Chong, T. H. Pohlman, C. R. Hampton, A. Shimamoto, N. Mackman, and E. D. Verrier Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury Ann. Thorac. Surg., February 1, 2003; 75(2): S649 - 655. [Abstract] [Full Text] [PDF]
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F. J. Schoen and R. F. Padera Jr. Cardiac Surgical Pathology Card. Surg. Adult, January 1, 2003; 2(2003): 119 - 185. [Full Text]
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R. M. Mentzer Jr., M. S. Jahania, and R. D. Lasley Myocardial Protection Card. Surg. Adult, January 1, 2003; 2(2003): 413 - 438. [Full Text]
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P. A. Poole-Wilson Innovative treatments for myocardial infarction; design, purpose and consequences of early studies Eur. Heart J., November 1, 2002; 23(21): 1640 - 1642. [Full Text] [PDF]
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Q.-D. Wang, J. Pernow, P.-O. Sjoquist, and L. Ryden Pharmacological possibilities for protection against myocardial reperfusion injury Cardiovasc Res, July 1, 2002; 55(1): 25 - 37. [Abstract] [Full Text] [PDF]
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S. Verma, P. W.M. Fedak, R. D. Weisel, J. Butany, V. Rao, A. Maitland, R.-K. Li, B. Dhillon, and T. M. Yau Fundamentals of Reperfusion Injury for the Clinical Cardiologist Circulation, May 21, 2002; 105(20): 2332 - 2336. [Full Text] [PDF]
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G. A Ortolano, G. S Aldea, K. Lilly, P. O'Gara, J. D Alkon, F. Madera, T. Murad, C. P Altenbern, C. S Tritt, A. Capetandes, et al. A review of leukofiltration in cardiac surgery: the time course of reperfusion injury may facilitate study design of anti-inflammatory effects Perfusion, March 1, 2002; 17(2_suppl): 53 - 62. [Abstract] [PDF]
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S. F. Khuri Evidence, sources, and assessment of injury during and following cardiac surgery Ann. Thorac. Surg., December 1, 2001; 72(6): S2205 - 2207. [Full Text] [PDF]
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T.-l. Yue, J. Chen, W. Bao, P. K. Narayanan, A. Bril, W. Jiang, P. G. Lysko, J.-L. Gu, R. Boyce, D. M. Zimmerman, et al. In Vivo Myocardial Protection From Ischemia/Reperfusion Injury by the Peroxisome Proliferator-Activated Receptor-{gamma} Agonist Rosiglitazone Circulation, November 20, 2001; 104(21): 2588 - 2594. [Abstract] [Full Text] [PDF]
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R. N. M. Cornelussen, G. J. Van Der Vusse, T. H. M. Roemen, and L. H. E. H. Snoeckx Heat pretreatment differentially affects cardiac fatty acid accumulation during ischemia and postischemic reperfusion Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1736 - H1743. [Abstract] [Full Text] [PDF]
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L. G. Melo, R. Agrawal, L. Zhang, M. Rezvani, A. A. Mangi, A. Ehsan, D. P. Griese, G. Dell'Acqua, M. J. Mann, J. Oyama, et al. Gene Therapy Strategy for Long-Term Myocardial Protection Using Adeno-Associated Virus-Mediated Delivery of Heme Oxygenase Gene Circulation, February 5, 2002; 105(5): 602 - 607. [Abstract] [Full Text] [PDF]
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