Endothelial Cell Injury in Cardiovascular Surgery: The Systemic Inflammatory Response1

Recent discoveries in the field of vascular biology have led to an expanded understanding of the pathogenesis of many of the immediate and long-term complications of patients undergoing cardiovascular operations and interventional cardiologic procedures. In particular, the vascular endothelium has emerged as the central focus of many of the biologic events that affect the preoperative, operative, and postoperative course of nearly all heart surgery patients. A recurring theme in the study of endothelial cell biology is the crucial role that endothelial cell injury plays in the difficulties that our patients encounter. The deleterious effects of endothelial cell injury are most evident in the acute syndromes of vasospasm, coagulopathy, ischemia/reperfusion injury, and the systemic inflammatory response to cardiopulmonary bypass. In addition, chronic endothelial cell injury contributes to the development of anastomotic narrowing and the progression of atherosclerosis, both of which limit the long-term success of coronary artery bypass grafting. Because of the increasingly recognized role of the endothelium in cardiovascular function there is a tremendous amount of basic science information detailing the response of the endothelium to injury. This is the fifth in a series of seven reviews intended as an introduction to the major topics of endothelial cell biology that are of importance to the practicing cardiothoracic surgeon. In particular, the authors have focused on the role that the endothelium has on the development of vasomotor dysfunction, bleeding and thrombosis, neutrophil-endothelial cell interaction, and obstructive arteriopathy. The aim of these reviews is to provide a concise reference point for cardiothoracic surgeons as they evaluate the ever-accumulating research findings and new therapies that stem from the study of the endothelium in response to the insults encountered in cardiothoracic surgery. Edward D. Verrier, MD

Endothelial Cell Injury in Cardiovascular Surgery: The Systemic Inflammatory Response Edward M. Boyle, Jr, MD, Timothy H. Pohlman, MD, Marion C. Johnson, MD, and Edward D. Verrier, MD Division of Cardiothoracic Surgery, University of Washington, Seattle, Washington

Many of the components currently used to perform cardiovascular operations lead to systemic insults that result from cardiopulmonary bypass circuit-induced contact activation, circulatory shock, and resuscitation, and a syndrome similar to endotoxemia. Experimental observations have demonstrated that these events have profound effects on activating endothelial cells to recruit neutrophils from the circulation. Once adherent to the endothelium, neutrophils release cytotoxic proteases and oxygenderived free radicals, which are responsible for much of

Inflammation in itself is not to be considered as a disease . . . and in disease, where it can alter the diseased mode of action, it likewise leads to a cure; but where it cannot accomplish that salutary purpose, . . . it does mischief. John Hunter: Treatise on the Blood, Inflammation, and Gunshot Wounds, London, 1794

T

he English surgeon John Hunter first recognized the malignant systemic spread of inflammation as an abnormal response to injury two centuries ago. The early pioneers in cardiac surgery recognized a similar pattern

Address reprint requests to Dr Verrier, Division of Cardiothoracic Surgery, University of Washington, 1959 Pacific St NE, Box 356310, Seattle, WA 98195 (e-mail: [email protected]).

© 1997 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

the end-organ damage seen after cardiovascular operations. Recently the cellular and molecular mechanisms of endothelial cell activation have become increasingly understood. It is conceivable that once the molecular mechanisms of endothelial cell activation are better defined, therapies will be developed allowing the selective or collective inhibition of vascular endothelial activation during the perioperative period. (Ann Thorac Surg 1997;63:277– 84) © 1997 by The Society of Thoracic Surgeons

of systemic injury they encountered after cardiopulmonary bypass (CPB). Kirklin [1] hypothesized that the deleterious effects of CPB were secondary to the exposure of blood to abnormal surfaces in the bypass circuit, which initiated a “whole body inflammatory response.” He noted that this response is characterized by activation of coagulation, the kallikrein system, fibrinolysis, and complement, all of which are now recognized as the mediators of the disseminated intravascular post-pump syndrome [1, 2]. Further work has identified the presence of circulating inflammatory cytokines, also observed in the systemic inflammatory response syndromes associated with shock and sepsis [3, 4]. Experimental observations have demonstrated that these cytokines have a profound effect on activating endothelial cells to partici0003-4975/97/$17.00 PII S0003-4975(96)01061-2

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pate in the inflammatory response to injury [5]. The end result of the humoral cascading that is initiated by CPB includes widespread endothelial cell activation, which in turn, likely results in the diffuse expression of leukocyte adhesion molecules on surfaces of vascular endothelial cells. Once adherent to the endothelium, neutrophils release cytotoxic proteases and oxygen-derived free radicals that are responsible for much of the end-organ damage seen after cardiac operations. Further suggestion of neutrophil-mediated injury, which occurs not only from complement activation but from increased neutrophil-endothelial adhesion, comes from studies that demonstrate neutrophil-derived proteases in the circulation after CPB. These proteases break down elastin, collagen, and fibronectin, destroying extracellular structures, and contribute to the capillary leak that leads to extracellular volume overload and electrolyte imbalance in the postoperative period [6]. Although it is difficult to demonstrate in studies with small patient populations, there seems to be a correlation between high neutrophil degranulation products and systemic complement activation and multiple system organ failure after CPB [6]. In addition to the inflammatory interactions that result from the CPB circuit, cardiogenic shock can further contribute to inflammatory response of cardiac operations. Some patients experience varying degrees of cardiogenic shock during CPB, when coming off the pump, and when recovering from the operation. Prolonged nonpulsatile perfusion or periods of circulatory arrest can lead to diffuse end-organ ischemia as well [7]. The end organ hypoxic insult likely causes endothelial cells, circulating monocytes, and tissue-fixed macrophages to release cytokines and oxygen-derived free radicals that drive this response. Once the patient is resuscitated from shock and after hypoxic end organs are reperfused, a form of systemic ischemia-reperfusion injury results [8]. Still another form of inflammatory activation that results from extracorporeal circulation and episodes of systemic ischemia-reperfusion is endotoxemia (lipopolysaccharide). Endotoxin is frequently detected in high concentrations in the systemic circulation after CPB [9, 10]. Endotoxin is a potent stimulant not only of complement activation, but of endothelial cell activation resulting in the surface up-regulation of adherence molecules and tissue factor [11, 12]. Endotoxin is a potent agonist of macrophage tumor necrosis factor release, which may explain why the level of this cytokine is elevated in some patients after CPB. Although the mechanism of endotoxemia after CPB is unclear, this may derive from a translocation of bacteria from the gut, resulting from the systemic stress of CPB and splanchnic ischemia, coupled with impaired Kupffer cell function [13]. The result is a transient endotoxemia that contributes to the overall state of systemic inflammation after CPB. Evidence that complement and cytokine cascades are involved in the pathologic inflammatory response to CPB has been reported in a number of studies that detail the presence and time course of circulating inflammatory elements as they appear in the bloodstream after the initiation of CPB. The most prominent early response is

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massive complement activation, heralded by a rapid increase in C5a and C3a, which appear in the blood that exits the extracorporeal circuit [2, 14]. C5a is a soluble by-product of complement activation. It serves as a marker for generalized complement activation, and itself causes capillary leak, neutrophil degranulation, and the expression of the neutrophil adhesion molecule Pselectin on the surfaces of platelets and the endothelium [15–17]. Cytokine release is also a prominent feature of the inflammatory response to CPB. Numerous investigators have detailed the presence and time course of circulating cytokines as they are released in response to CPB. Once bypass is initiated, levels of interleukin-1 (IL-1), tumor necrosis factor, IL-6, and IL-8 rapidly increase [18 –22]. The degree of cytokine response appears to correlate with the length of CPB and aortic cross-clamp time [23]. What is largely unknown, however, is where these cytokines originate. Experiments using simulated extracorporeal circuits have demonstrated that complement is activated in the bypass circuit; however, the cytokine response does not resemble what is seen clinically [14]. Neutrophils, macrophages, and endothelial cells in culture release cytokines in response to injury [24, 25]. It is possible that the cytokine response results from activated endothelial cells, adherent neutrophils, and tissue-fixed macrophages, driven by complement activation and myocardial and systemic ischemia-reperfusion injury during the course of the operation. The fact that many patients successfully recover from CPB, despite this massive inflammatory response, suggests that the individual responses to inflammatory stimuli vary from person to person. Furthermore, it attests to the body’s tremendous physiologic reserve and sophisticated inhibitory pathways that prevent widespread organ damage after heart operations [1]. Recently, however, an increasing number of patients undergoing cardiac operations have a limited physiologic reserve. Neonates and infants as well as the elderly and those who require long CPB times are especially susceptible to systemic effects of endothelial cell injury [1]. As patients undergo operations in a worsening physiologic state it is increasingly important to seek improved understanding of the biological mechanisms of organ damage and new therapies to modulate the injury suffered after cardiac operations.

Systemic Endothelial Cell Activation The vascular endothelium has a pivotal role in the systemic host response to injury and therefore, the systemic injury that follows CPB [26]. Central to the understanding of the endothelial injury after cardiac operation is the concept of endothelial cell activation. Under resting conditions the endothelial cell lining of blood vessels is a relatively inert surface that regulates the passage of intravascular substrates to the extravascular space and assures the unhindered flow of cellular and serum elements through the capillary beds. In response to inflammatory signals, such as cytokines, lipopolysaccharide, complement activation products (C5a), hypoxia, or oxygen-derived free radicals, endothelial cells are converted

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to an activated state, resulting in profound changes in gene expression and cellular function. Activated endothelial cells release cytokines and express proteins on their surface that promote inflammatory reactions and thrombosis [12, 27]. In the classic host response to injury, endothelial cell activation is beneficial in recruiting neutrophils and promoting coagulation to limit the local spread of infection. Although this response is extremely destructive, the limited area of collateral tissue injury is usually well tolerated on the local level. The host response is less well adapted to respond to injury on a systemic level. Cardiopulmonary bypass and other components of modern critical care have led to survival despite severe systemic illness and physiologic stresses that the human body probably never evolved to control. Therefore, after CPB, sepsis, or resuscitation from shock, well evolved local inflammatory responses to injury become manifested systemically with the release of cytokines, the activation of large areas of endothelium, and the margination and degranulation of neutrophils on a much larger scale. The feedback mechanisms that limit the destructive response locally appear to be less accurate on a systemic level, leading to an ineffective and overwhelming host response. This overwhelming response is evident after CPB, where evidence suggests that the activated vascular endothelial layer promotes widespread leukocyte adhesion molecule and tissue factor expression, mediating end-organ damage and consumption of coagulation factors that contributes to the coagulopathy that often complicates cardiac operations [28 –33]. This is also evident in other syndromes of systemic inflammation, such as systemic inflammatory response syndrome, adult respiratory distress syndrome, and multiple system organ failure, where the host’s response to injury actually contributes to further organ injury [3, 4, 24, 34]. Therefore, understanding the signals that result in endothelial activation is important in assessing potential therapies to block this response. The cellular and molecular mechanisms of endothelial cell activation have been elucidated in endothelial cells cultured from human umbilical veins [5]. The process of endothelial cell activation involves a complex series of cellular and molecular steps, each step representing a potential point of therapeutic control. There appears to be two phases of endothelial cell activation that contribute to organ dysfunction after CPB. In the first, the immediate phase, circulating complement degradation products initiate an immediate but short-lived neutrophil adhesive response. The second phase requires several hours to develop as new proteins, such as E-selectin, ICAM, and IL-8, are made and expressed by endothelial cell in response to exposure to circulating cytokines, lipopolysaccharide, and inflammatory mediators [35–38] (Fig 1).

Complement Mediated Endothelial Cell Activation The first wave of inflammatory mediators released systemically after the initiation of CPB are the by-products

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of complement activation. Early studies demonstrated that complement activation plays a major role in contact activation secondary to CPB. There are a variety of components of a cardiac operation that potentially lead to systemic complement activation. The most studied and probably the most significant event is the interaction of blood with the CPB circuit [39]. How complement activation itself actually results in cellular damage after cardiac operation, however, was largely unknown. Recent improvements in understanding the mechanisms of complement-endothelial cell interactions has led to an increased appreciation of the role complement activation plays in contributing to the widespread endothelial cell activation that leads to end organ damage after CPB. Complement activation can occur through the classic or alternative pathways (Fig 2). The classic pathway requires the formation of an antigen–antibody complex, which occurs only briefly in cardiovascular operation. For example, classic pathway activation likely occurs in some patients in response to preformed antibodies that recognize some component of the heparin–protamine complex [40]. The alternative pathway, on the other hand, accounts for a majority of the complement activation that occurs in response to CPB. The alternative system is constantly laying down C3b on all surfaces with which it comes in contact. Those surfaces that possess the inhibitory proteins, such as decay accelerating factor, immediately destabilize the C3b and prevent amplification of the process. In this way the alternative pathway distinguishes self from nonself [41]. When C3b is deposited on surfaces that lack the inhibitory proteins to stop the process, those surfaces serve as the staging ground for further activation resulting in formation of the membrane attack complex (C5b-9) and the generation of anaphylatoxins (C5a and C3a). When blood is run through the pump the exposure of the foreign surfaces to serum complement proteins results in C3b being laid down on the circuit surfaces. This, in turn, results in a geometric amplification of the alternative pathway of complement and the release of large quantities of C5b-9, C3a, and C5a into the circulation. Thus, the actual complement activation occurs in the conduits and components of the pump; however, the damaging effects occur remotely when the by-products of complement activation (C5a and C3a) return to the circulation. In 1981 Chenoweth and colleagues [2] detailed the immense complement activation that follows bloodartificial surface interactions in the pump oxygenator. Specifically, they showed that when CPB was initiated there was a rapid increase in C3a and C5a that coincided with a swift decrease in white blood cell levels secondary to sequestration in the lungs. At first it appeared that many of the damaging effects of complement activation could be attributed to elevated levels of C3a. Subsequent studies have failed to prove this correlation and the congenital absence of C3 fails to offer the degree of protection that would be expected [30, 42]. C5a, however, has emerged as an important complement component in the resulting damage that follows CPB. The relationship

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Fig 1. The disseminated intravascular postpump syndrome. (ICAM 5 intracellular adhesion molecule; IL 5 interleukin; TNF 5 tumor necrosis factor; VCAM 5 vascular cell adhesion molecule.)

between circulating C5a and the pulmonary and other end organ leukosequestration that Chenoweth and colleagues demonstrated occurred during CPB has recently been linked as causal. Studies by Foreman and colleagues [17] demonstrate that C5a is a potent agonist of endothelial cell P-selectin surface expression. These in vitro observations of complement/endothelial interactions suggest that P-selectin, expressed as a result of endothelial cell C5a exposure, could mediate the sudden leukosequestration that follows the initiation of CPB. Once adherent to the complement activated endothelium, neutrophils can release proteases, oxygen-derived free radicals that contribute to endothelial cell barrier dysfunction and fluid extravasation. This hypothesis is consistent with the fact that P-selectin is unique, compared to other adhesion molecules, because it is the most readily available of the adhesion molecules. It is stored

ready made in cytoplasmic vacuoles (the Weibel Palade bodies), rapidly reaching the plasma membrane by exocytosis after endothelial activation [43]. Once expressed on the lumenal surface, P-selectin is available to recruit neutrophils from the passing circulation. Therefore, this rapid response phase results in an almost immediate adhesion of neutrophils to the microvasculature systemically, where the neutrophils are free to release proteases and oxygen-derived free radicals that directly lead to diffuse capillary leak, impaired oxygenation, and a host of other detriments already described. Furthermore, C5a activates neutrophils to express adhesive properties such as CD11B/CD18 (Mac-1), which can bind with ICAM-1 on activated endothelial cells [44]. C5a activates platelets and monocytes, resulting in the release of cytokines and other inflammatory mediators that amplify neutrophilendothelial cell adhesion. In addition to its direct actions

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Fig 2. The classic and alternative pathways of complement activation. The classic pathway requires an antigen-antibody complex to be formed. This occasionally happens when protamine is given to reverse heparin. The alternative pathway is nonspecific, and can be activated by foreign surfaces and the surface of endotoxin. When complement is activated, soluble factors, such as C5a and C3a, are released into the circulation.

in activating endothelial cells, neutrophils, monocytes, and platelets, activated complement fragments significantly augment the neutrophil adhesive response by greatly increasing the sensitivity of endothelial cells to inflammatory mediators such as tumor necrosis factor [45].

Prolonged Adhesion Molecule Expression Although it is likely that complement activation mediates the first and most immediate wave of leukocyte adherence molecule expression, both in vitro and in vivo studies reveal that this response is short lived [33, 46]. As the early phase of complement-mediated neutrophil adhesion wears off, a second wave develops as the other mediators of inflammation reach peak levels over the ensuing hours. Cytokine levels proportionately increase in direct response to the length of the procedure, activating endothelial cells throughout the body. Inflammatory

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mediators, such as cytokines or lipopolysaccharide, bind to specific receptors on the surface of the endothelium and stimulate the resting endothelium to convert to the activation state. Signals are transmitted through the cell wall, through a wide variety of second messenger systems, to signal transduction pathways that translocate to the nucleus to turn on a specific set of genes, known as the activation genes [47]. There are multiple stretches of DNA that make up the activation genes, which are transcribed as a result of endothelial cell activation. In the patient undergoing cardiovascular operation, the most significant are the genes that encode for E-selectin, ICAM, VCAM, IL-8, inducible nitric oxide synthase, and tissue factor. A tremendous amount of information has recently emerged that details the molecular signaling pathways that lead to the transduction of extracellular signals to the regulatory regions controlling the genes that are activated in CPB patients. What is most notable is that this wide array of inflammatory signals appear to funnel through a single signal transduction pathway using the transcription factor NF-kB [48, 49]. NF-kB is a ubiquitous transcription factor involved in the regulation of genes that respond to various forms of external stimulation [47] (Fig 3). Once activated, NF-kB translocates to the nucleus where it binds with specific DNA sequences, altering conformation of the basal transcriptional apparatus, resulting in the transcription of the various activation genes. Usually NF-kB is bound to IkB, the cytosolic inhibitory protein that keeps NF-kB inactive [50]. When activated by cytokines, lipopolysaccharide, or hypoxiareoxygenation, the NF-kB-IkB complex is phosphorylated, and the complex becomes dissociated. Once dissociated, IkB is degraded rapidly, and in parallel, there is an accumulation of NF-kB in the nucleus. This results in the initiation of transcription of the activation genes. The DNA of the activation genes transcribe specific message RNA transcripts that are then translated into proteins in

Fig 3. Signal transduction through NF-kB. (ICAM 5 intracellular adhesion molecule; IL-1 5 interleukin-1; LPS 5 lipopolysaccharide; TNF 5 tumor necrosis factor.)

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the cytoplasm, modified, and later expressed on the cell surface or released into the local environment. This process takes about 4 hours and peaks at 8 to 24 hours, depending on the gene [36, 37]. In addition, when endothelial cells are activated, transcription of IkB is promoted, which feeds back to bind with NF-kB and thereby decrease levels of free NF-kB, subsequently shutting off expression of the NF-kB activated genes [50]. Thus, the release of IkB appears to be the central event required for the activation of NF-kB, and ultimately, for gene activation and syntheses of new proteins in response to extracellular stimuli. There are several principal endothelial cell activation proteins that are expressed after CPB in this manner. The first 4 to 8 hours after CPB, E-selectin, ICAM, and IL-8 are manufactured in endothelial cells. These molecules take several hours before they are expressed on the surface (E-selectin, ICAM) or released (IL-8) because they require de novo protein synthesis. E-selectin mediates the initial rolling of the leukocyte through low affinity binding, ICAM forms the firm bond, and IL-8 activates neutrophils and facilitates transendothelial cell migration to the underlying tissue where the neutrophil does its damage. The important role of these molecules after CPB is supported by a recent study by Kilbridge and colleagues [31] who demonstrated the induction of ICAM-1 and E-selectin in the hearts and skeletal muscle of pediatric patients after CPB. Therefore, once the first wave of adherence molecules, P-selectin, wears down, the secondary wave of adhesion molecules are upregulated (E-selectin, ICAM), leading to a more prolonged leukocyte-endothelial cell attachment, with the resulting destructive consequences [31, 33]. Because of the transient nature of endothelial cell activation, most endothelial cells loose their adhesive properties shortly after the cytokine and C5a levels return to baseline. After most cardiac operations, in the absence of any secondary episodes of shock or infection, this should be within 24 to 48 hours.

Potential Therapeutic Options Techniques designed to impair neutrophil-endothelial cell interaction have the potential to attenuate the whole body inflammatory response to CPB. Having an impact on neutrophil-endothelial cell interaction can occur in a variety of ways. Because a majority of the inciting events begin as a result of the blood’s interaction with the CPB circuit, efforts have been focused at altering the components of the extracorporeal oxygenator. The bubble oxygenator, especially when containing nylon mesh, was shown to result in a greater degree of complement activation and pulmonary leukosequestration prompting many surgeons to turn to membrane oxygenators [2, 51, 52]. Advances in biomedical engineering have led to the ability to coat CPB lines with heparin, which seems to increase the biocompatibility of the circuit and decrease complement activation [53–55]. Another available device is the Pall filter, which allows the removal of neutrophils from the circulation, decreasing their availability to pro-

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mote inflammatory injury [52, 56]. Unfortunately leukocytes return to the circulation when the filter is discontinued. Although these techniques may appear helpful, their utility in clinical practice are as yet unproven [57]. There are a variety of additional techniques available to block neutrophil adhesion to the activated endothelium. Specific monoclonal antibodies were developed by cellular biologists as tools to identify adhesion molecules expressed on cytokine-activated endothelium. The exploitation of this advance as a therapeutic tool led not only to an improved understanding of the role of adhesion molecules but also to novel avenues to modulate this response. Today there are an increasing number of monoclonal antibodies available to block adhesion molecules before neutrophils can adhere. The objective of antiadhesion therapy after CPB should be to prevent neutrophil adherence during the first 24 hours after operation, thereby preventing the neutrophils from mediating widespread organ damage. In designing therapies to block this critical leukocyte-endothelial interaction, it is imperative to address the time courses of expression of specific endothelial adherence molecules so that interventions can be directed to each specific adhesion molecule at the time when it can be expected to be maximally expressed. Monoclonal antibodies blocking selectins (E-selectin, P-selectin), or circulating oligosaccharide antagonists that block the interaction of the neutrophil S lex ligand with endothelial selectins, prevent the rolling and subsequent adherence of neutrophils. Gillinov and associates [28] used the antiinflammatory agent NPC 15669 to inhibit neutrophil adhesion in a CPB model and found a marked decrease in pulmonary injury. Adhesion molecule blockade, however, increases susceptibility to infection, limiting that approach as a therapeutic strategy [58]. Furthermore, once the endothelial cell is activated it expresses a broad variety of surface adhesion proteins at different time intervals that would require a complex mixture of agents to effectively block the consequences of endothelial activation. Rather than blocking the surface proteins after they are expressed, an alternative approach would be to block activation before adhesion molecules are made by endothelial cells. One of the simplest ways to block cellular machinery is with hypothermia. The time-tested technique of hypothermia is basically an attempt to shut down the cellular metabolic activity during ischemic cardiac arrest. The effects of hypothermia on endothelial function, however, have been minimally studied [59]. We investigated the effect of hypothermia on endothelial cell activation and found that although activation was temporarily halted below 25°C, as evidenced by E-selectin and tissue factor surface expression, NF-kB still accumulates in the nucleus until the cells rewarm, when transcription begins and activation continues unabated [60]. These findings have been corroborated clinically by Menasche and colleagues [61, 62] who found that hypothermia delays but does not prevent the expression of neutrophil adherence molecules. Therefore, although hy-

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pothermia is beneficial when the patient is cold, the benefits are rapidly lost when the patient rewarms. Investigators are now examining the molecular signaling processes that regulate expression of individual adhesion molecules, many of which share similar signal transduction pathways. Studies of the molecular mechanisms promoting the expression of endothelial cell activation genes are of particular interest in the development of novel therapies to attenuate the organ dysfunction and coagulopathy sometimes seen after cardiac operations. Efforts to further characterize the molecular events that result endothelial cell activation after inflammation may allow the utilization of the growing number of genedirected techniques to modulate and thereby prevent some of the inflammation and coagulopathy that frequently complicates cardiovascular operations. It is conceivable that once the molecular mechanisms of endothelial cell activation are better understood, therapies will be developed that will allow the selective or collective inhibition of vascular endothelial activation during the perioperative period, allowing patients to better tolerate cardiac operations.

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