Pathophysiology 5 (1998) 167 – 184
The role of leukocyte-endothelial cell adhesion in cardiovascular disease Michael J. Eppihimer * Genetics Institute, Preclinical Research and De6elopment, Pharmacology Research, 1 Burtt Road, Ando6er, MA 01810, USA Accepted 25 September 1998
Abstract The accumulation of leukocytes in inflamed tissue results from a series of adhesive interactions between leukocytes and endothelial cells in the microvasculature. It is recognized that leukocyte adhesion to vascular endothelium is an early and rate-limiting step in the leukocyte infiltration and tissue injury associated with acute and chronic diseases of the cardiovascular system. The magnitude of leukocyte-endothelial cell adhesion is determined by an array of factors including: the expression of adhesion molecules on the surfaces of leukocytes and endothelial cells, the inflammatory mediators which are secreted by activated leukocytes and endothelial cells and the hemodynamic forces resident within the blood vessels. In this review, the adhesion molecules that participate in the recruitment of leukocytes are described and discussed in terms of their relevance to the pathogenesis of cardiovascular disease. In addition, the pro- and anti-adhesive cellular products that mediate leukocyte-endothelial cell adhesion in ischemia and reperfusion injury (i.e. myocardial and cerebral) and atherosclerosis will be addressed. Finally, the significance of neutralizing the adhesive interactions between leukocytes and endothelial cells is discussed as a strategy for treating cardiovascular disease. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inflammation; Adhesion; Selectins; Integrins; Reperfusion; Atherosclerosis
1. Introduction Leukocyte adhesion to vascular endothelium is a classical response in many cardiovascular disorders. In recent years, many studies have been designed to address the cellular and molecular mechanisms involved with mediating the interactions between leukocytes and endothelial cells. Several factors appear to contribute to leukocyte-endothelial cell adhesion these include: (1) the magnitude of adhesion molecule expression on leukocyte and/or endothelial cell surfaces (2) the release of inflammatory mediators by leukocytes, endothelial
* Tel.: +1 978 6231597; fax: + 1 978 6231333; e-mail: [email protected]
cells, macrophages, etc. and (3) the hemodynamic dispersal forces that act to sweep leukocytes from the microvessel wall. The development of monoclonal antibodies and gene-targeted mice aimed at neutralizing adhesive interactions have become powerful tools in examining the consequences of interfering with leukocyte-endothelial cell adhesion in the progression of many diseases. In this review, the available evidence in the literature concerning the role of leukocyte-endothelial cell adhesion in mediating vascular dysfunction and tissue necrosis in cardiovascular pathologies will be examined. Specifically, the cell adhesion molecules associated with myocardial infarction, stroke, and atherosclerosis will be addressed in terms of their expression on leukocytes and endothelial cells, their involvement in leukocyte recruitment and the therapeutic benefits of blocking their function.
0928-4680/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0928-4680(98)00023-6
M.J. Eppihimer / Pathophysiology 5 (1998) 167–184
2. Selectin family of endothelial cell surface molecules
The emergence of leukocytes from the vascular to the interstitial compartment requires that the leukocyte come in contact with the blood vessel wall. For initial contact to occur, leukocytes must be displaced from the blood stream towards the periphery of the vessel, a process which is mediated by the radial dispersal forces within the blood vessel . Upon exiting the capillaries, the more deformable red blood cells which pile up behind the leukocytes in the capillary segments overtake the leukocytes and deflect them towards the venular wall, thereby increasing the opportunity for the leukocytes to initiate contact with the venular endothelium. In vivo observations suggest that this process, called margination, requires the interactions between red blood cells and leukocytes [2,3]. Following their margination, leukocytes undergo a series of adhesive interactions which begin as a rolling movement along venular endothelium and is succeeded by stationary adhesion and subsequently, extravasation across the vessel wall. Leukocyte rolling is a prerequisite for firm adhesion, as evidenced by a positive correlation of the level of leukocyte rolling present in venules to the level of firm adhesion in venules . This is further substantiated by studies demonstrating that administration of monoclonal antibodies (mAbs) directed against the selectins (which mediate leukocyte rolling, see below) attenuates the recruitment of adherent leukocytes in inflamed venules [5– 7,9]. Leukocyte rolling is often described as a low affinity adhesive interaction between leukocytes and endothelial cell, which is induced by the force of flowing blood acting on the leukocyte to cause its rotational movement. Leukocyte rolling is mediated by a distinct family of adhesion molecules residing on both endothelial cells and leukocytes, known as the selectin family of cell adhesion receptors. This family includes E- and P-selectin, which are expressed on the surface of stimulated endothelial cells, with the latter also found on the surface of activated platelets [10,11]. L-selectin is constitutively expressed on the surface of all leukocytes and is shed by proteolysis upon activation . The three selectins are structurally similar carbohydrate-binding lectins, consisting of an n-terminal lectin domain, an epidermal growth factor domain and a series of consensus repeats similar to those in complement proteins . Several leukocyte receptor(s) have been identified as counter ligands for E- and P-selectin. Sialyl – lewis X and other fucosylated carbohydrates  and P-selectin glycoprotein ligand-1 (PSGL-1, ) interact with Eand P-selectin to mediate leukocyte adhesion. Studies have also found that PSGL-1 may act as ligand to L-selectin during leukocyte-leukocyte interactions . Finally, using intravital microscopic analysis, Kubes et al.  revealed that L-selectin may also act as a ligand to P-selectin.
P-selectin, the largest of the selectin adhesion molecules, is stored in alpha-granules of platelets and Weibal Palade bodies of vascular endothelial cells [11,13,18]. Cloning data from an endothelial cell library predicts that multiple forms of P-selectin exist, one of which lacks a transmembrane and cytoplasmic domain . Platelets contain approximately equal amounts of P-selectin mRNA endcoding the protein with and without the transmembrane and cytoplasmic tail . While in vitro studies suggest that little or no P-selectin is expressed on unstimulated endothelial cells [11,20], recent in vivo studies have demonstrated that significant levels of constitutive P-selectin expression may exist in tissues such as the small intestine and the lung . P-selectin is unique among the selectin family of molecules since its expression on the surface of platelets and endothelial cells may be rapidly upregulated from storage granules following activation with agents such as histamine and thrombin . In vitro analysis revealed that histamine induces P-selectin expression to the endothelial cell surface within 5 min, with its reinternalization occurring by 20 min . These elevations in P-selectin expression coincide with increases in the number of leukocytes rolling along the endothelium, a response that is inhibited by treatment with a P-selectin specific mAb . In the mouse, it was observed that P-selectin expression reaches maximal values 10–30 min after histamine stimulation, however, unlike in vitro studies, P-selectin expression remains significantly elevated up to 60 min after stimulation . In addition, histamine-induced P-selectin expression is H1-receptor dependent and H2-receptor independent, as evidenced by the ability of diphenhydramine, but not cimetidine, to prevent the histamine-induced increase in P-selectin expression . These data agree favorably with intravital microscopic observations which revealed that histamine-induced leukocyte rolling in postcapillary venules is mediated through the H1-receptor . Oxygen radicals may also induce P-selectin expression on the surface of endothelial cells, but with kinetics that are markedly different from induction with histamine and thrombin . Oxidants produce a timedependent increase in P-selectin expression with peak expression occurring 2 h after stimulation and remaining elevated up to 4 h. These elevations in P-selectin expression were paralleled with an increase in neutrophil adhesion . Given that von Willebrand Factor (vWF) was observed to be elevated in the supernatant, confirms that the Weibel–Palade bodies fused with the plasma membrane and released their contents. This strongly suggests that the upregulation of P-selectin following oxidant stimulation is due to its translocation from cellular storage vesicles.
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In addition, P-selectin may be synthesized in endothelial cells through a transcriptional mechanism activated by endotoxin or tumor necrosis factor-a (TNF-a [26,27]). In vitro and in vivo studies demonstrate that P-selectin reaches peak expression 3 – 4 h after cytokine stimulation [21,26,28]. The rise in P-selectin expression coincides with elevations in P-selectin mRNA, supporting a de novo synthesis pathway for P-selectin . Following peak expression, P-selectin declines to baseline values in vitro  but remains significantly elevated in vivo , suggesting that other inflammatory cells such as macrophages may contribute to the sustained expression of P-selectin. Furthermore, the de novo synthesis of P-selectin functions in parallel with (and independent of) the rapidly induced mobilization of P-selectin from storage granules . While endothelial cell surface membrane-associated P-selectin promotes leukocyte adhesion, the soluble form of P-selectin (lacking the transmembrane and cytoplasmic domain) has been proposed as an anti-adhesive molecule. Pretreatment of TNF-a stimulated neutrophils with soluble P-selectin inhibits their adhesion to resting endothelial cell monolayers in a dose-dependent manner . However, pretreatment of activated endothelium with soluble P-selectin was ineffective in blocking neutrophil adhesion, suggesting that the anti-adhesive function of soluble P-selectin is due to its binding to the neutrophil surface . In addition, soluble P-selectin inhibits the capacity of neutrophils to generate superoxide anion . Under normal conditions soluble P-selectin is found in circulating plasma  and is elevated in a number of cardiovascular pathologies [32–35] suggesting that soluble P-selectin levels may reflect the magnitude of endothelial cell and/or platelet activation.
2.2. E-selectin Unlike P-selectin, there are no preformed pools of E-selectin, but requires de novo synthesis following activation with cytokines or endotoxin [10,36]. Under basal conditions, E-selectin is not present on the surface of endothelial cells in vivo and in vitro [21,36 – 38]. In vitro and in vivo studies have demonstrated that E-selectin is upregulated within 2 h, reaches maximal levels by 3 –4 h and returns to baseline values at 24 h after endothelial cell activation [21,28,36,39]. These elevations in the glycoprotein coincided with measurements of elevations of E-selectin transcript . Antibodies-directed against E-selectin are effective in attenuating the rolling, adhesion and transmigration of neutrophils at these peak times of E-selectin expression [37,40,41]. At comparable time points in vivo, leukocyte rolling was also attenuated in cytokine-treated venules after the administration of a mAb directed against E-selectin . In other in vivo studies, the time course of leuko-
cyte recruitment was monitored during measurements of E-selectin expression . These studies demonstrate that infiltration of leukocytes parallels the upregulation of E-selectin expression. While these findings implicate a role for E-selectin in mediating leukocyte-endothelial cell interactions, numerous studies have been unable to show a definitive role for E-selectin as a mediator of leukocyte rolling [44,45]. A possible explanation for a lack of efficacy of E-selectin mAbs to attenuate leukocyte rolling in inflamed venules is the redundancy in function between E- and P-selectin to mediate leukocyte rolling. In E-selectin knockout mice, leukocyte emigration into the peritoneum was not significantly different from wild-type mice, but was significantly attenuated following administration of a P-selectin mAb . Assessment of the kinetics E- and P-selectin expression in mice revealed a similarity in the period and magnitude of upregulation, indicating that both adhesion molecules may need to be blocked to produce an attenuation in leukocyte rolling . Although soluble isoforms of E-selectin are found in supernatants taken from cytokine activated endothelial cells  and sera obtained from patients with cardiovascular pathologies [47,48], there is no evidence demonstrating that it is produced by alternative splicing, as observed with soluble P-selectin. The molecular weight of soluble E-selectin is in close agreement with predicted molecular weights of E-selectin cleaved at or close to the endothelial cell membrane . Although soluble P-selectin appears to have anti-adhesive properties, studies have indicated that soluble E-selectin may have cytokine-like proinflammatory effects. These effects include potentiating b2-integrin dependent adhesion of neutrophils, oxygen radical production and oxidative burst . In addition, soluble E-selectin has been demonstrated to induce chemotaxis of neutrophils . Given these apparent properties of soluble E-selectin on neutrophil function, it is possible that soluble E-selectin may exacerbate neutrophil-mediated vascular injury. However, the pro- and anti-inflammatory effects of soluble selectins in cardiovascular diseases warrants further attention.
3. Immunoglobulin superfamily of endothelial cell adhesion molecules
3.1. ICAM-1 Although both rolling and firmly adhering leukocytes are recruited into postcapillary venules of inflamed tissues, more emphasis has been placed on monitoring and quantifying the latter adhesive interactions. Emphasis has been placed on adherent leukocytes, rather than rolling leukocytes, since it is likely that firm adhesion of leukocytes in postcapillary venules plays an
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important role in the modulation of transendothelial emigration and possibly, microvascular permeability. Whether or not leukocytes firmly adhere to vascular endothelium depends on the balance between adhesive forces generated by the glycoproteins on leukocytes and endothelial cells, and the dispersal forces generated by the flowing blood [50,51]. Firm adhesion between leukocytes and endothelial cells is mediated by the supergene immunoglobulins located on the surface of endothelial cells and their ligands, the b2 integrins, located on most leukocytes . The two subunits of CD11/CD18, CD11a and CD11b, which have been proposed as ligands for ICAM-1, bind to different domains on ICAM-1 . Most in vivo and in vitro studies agree that a significant level of ICAM-1 is expressed on endothelial cells [20,36,38,53–55]. Similar to E-selectin, the expression of ICAM-1 is regulated through a transcriptional mechanism . In cell culture systems, ICAM-1 is maximally upregulated on the surface of the endothelial cells by 8 h and remains significantly elevated for 48 h in response to endotoxin, TNF-a and interleukin-1b (IL1b, ). In vivo studies have demonstrated that ICAM-1 is upregulated within 2 h of endothelial activation by TNF-a and with maximal levels occurring at 5 h after stimulation . Thereafter, the level of ICAM1 remained significantly elevated for 24 h. These observations of ICAM-1 on the surface of endothelial cells coincides with peak levels of ICAM-1 mRNA occurring 3 h after endothelial cell activation . In most tissues, the level of ICAM-1 mRNA had returned to basal levels by 9 h. Antibodies directed against ICAM-1 are more effective in attenuating the adhesion of neutrophils to cytokine-activated endothelium at 24 –48 h than at 4 h and is presumably due to the greater expression of ICAM-1 at these later times [37,40]. A soluble isoform of ICAM-1 has been identified in serum of healthy patients and is elevated in serum of patients during inflammatory conditions [56,57]. Although it was generally thought that the soluble isoform of ICAM-1 represented cleaved fragments of membrane-associated ICAM-1 [46,58], it has been demonstrated that human tissues possess mRNA that encodes for a soluble isoform of ICAM-1 . Recent studies provide evidence to support the alternative splicing of a soluble form of ICAM-1. In these studies, peak levels of TNF-a-induced soluble ICAM-1 occurred at 5 h compared to membrane-associated ICAM-1 levels, which peaked at 12 h, suggesting that there are different mechanisms in the synthesis of soluble and membrane-associated ICAM-1 . ICAM-1deficient mice displaying negligible levels of membrane-associated ICAM-1 were also found to have significant levels of soluble ICAM-1, which was in-
creased following TNF-a administration . Given that shedding membrane associated ICAM-1 is unlikely in ICAM-1 deficient mice, these observations indicate that spliced variants of ICAM-1 may contribute significantly to the circulating levels of ICAM-1. Regardless of the source of soluble ICAM-1, a physiological role for this protein was demonstrated in vitro whereby soluble ICAM-1 at concentrations of 200 ng/ml inhibited lymphocyte adhesion to cerebrovascular endothelial cells . Since circulating levels of ICAM-1 are : 200 and 600 ng/ml under basal and inflammatory conditions , respectively, these data suggest that soluble isoforms of ICAM-1 may be an important mechanism in regulating leukocyte-endothelial cell adhesion.
3.2. CD11 /CD18 complex The adhesion complex, CD11/CD18 is composed of three structurally and functionally related glycoprotein heterodimers each consisting of a distinct a subunit (CD11a, CD11b or CD11c) that is non-covalently associated with a common b subunit (CD18). CD11a/CD18 is expressed on virtually all leukocytes whereas CD11b/ CD18 is expressed on neutrophils, monocytes and macrophages . Both are constitutively expressed on the surface of non-activated leukocytes and are rapidly induced into a high avidity state following exposure of the leukocyte to inflammatory agents such as phorbal esters and formyl peptides . In addition, these agents may also elicit a translocation of CD11b/CD18 from intracellular storage granules to the leukocyte surface . For example, activation of neutrophils with f-Met-Leu-Phe (FMLP) induces a 6-fold increase in CD11b/CD18 expression on the cell surface . Monoclonal antibodies directed against CD11/CD18 have been useful in assessing the role of these adhesion glycoproteins in leukocyte-endothelial cell adhesion and leukocyte function. CD11b but not CD11a-specific mAbs are effective in inhibiting neutrophil aggregation induced by chemotactic factors and phorbal esters . Evidence in the literature that indicates that CD11a and CD11b are ligands for ICAM-1 [65–68]. Smith et al.  demonstrated that the adhesion of FMLP-activated neutrophils to endothelial cells is dependent on both CD11a and CD11b. A combination of mAbs to both CD11a and CD11b resulted in a cumulative inhibition of neutrophil adhesion, compared to each mAb alone . Furthermore, neutrophil adhesion to unstimulated endothelium is inhibited by a mAb directed against CD11a, and because ICAM-1 mAbs inhibit both unstimulated and stimulated neutrophil adhesion to endothelial cells, it was determined that both CD11a and CD11b bind to distinct sites on ICAM-1 .
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Fig. 1. Mechanism proposed to explain the accumulation of leukocytes in myocardial tissue exposed to ischemia and reperfusion. Elevation in ROMs, inflammatory mediators and complement activation induces an elevation in leukocyte and endothelial cell adhesion molecules, and consequently, an increase in leukocyte adhesion and emigration and tissue injury.
4. Myocardial ischemia and reperfusion injury Reperfusion of ischemic tissue plays an important role in the pathogenesis of many cardiovascular disorders including myocardial infarction, stroke, hemorrhagic shock and organ transplantation . Although vascular dysfunction following reperfusion is multi-factorial, a role for leukocytes in exacerbating tissue injury is provided by observations that neutrophil depletion attenuates postischemic cellular injury in the heart , brain , and small intestine . Since compounds that inhibit leukocyte adherence to postcapillary venules reduce the accumulation of leukocytes in postischemic tissue, and subsequent tissue injury, pharmacological inhibition of leukocyte-endothelial cell adhesion may be an important therapy prior to restoring blood flow to ischemic tissues. During myocardial reperfusion, a series of biochemical and cellular events occur which mediate vascular injury. In particular, an enhancement in the production of reactive oxygen metabolites (ROMs) , activation of complement [74,75], diminished nitric oxide production [8,76] and leukocyte adhesion to vascular endothe-
lium are associated with the reperfusion of ischemic myocardium [77,78] (Fig. 1). Additional evidence for leukocyte-mediated injury in the reperfused myocardium, is afforded by the observations of a reduction in myocardium necrosis in dogs receiving neutrophil antiserum . In studies using filters to render animals neutropenic, dogs subjected to 90 min of ischemia and 2 h of reperfusion were also found to have a significant reduction in myocardial infarct size . Sheridan et al.  also found that neutropenia attenuated the magnitude of vascular protein leakage following reperfusion, suggesting that leukocytes play an important role in endothelial cell dysfunction. Although these studies demonstrate a role for leukocytes in the pathogenesis of myocardial injury, the specific mechanisms whereby the leukocytes mediate the injury remains unclear. Given the availability of mAbs directed against leukocyte and endothelial cell adhesion molecules, the role of adhesive interactions in myocardial injury has been investigated extensively. Following a brief period of ischemia, P-selectin expression reaches peak values in myocardial venules at 20 min of reperfusion . Thereafter, P-selectin expression declined to-
M.J. Eppihimer / Pathophysiology 5 (1998) 167–184
ward baseline values during a 5 h reperfusion period. This study also demonstrated the presence of P-selectin in arterioles at 20 min after reperfusion, with the expression returning to baseline values within 60 min. In cats, a P-selectin antibody was observed to reduce myocardial necrosis by 55% and inhibit neutrophil adhesion to coronary artery segments subjected to I/R . These data suggest that immunoneutralization of early adhesive interactions (at 20 min of reperfusion) may provide protection to the myocardium. In addition, P-selectin mAbs were also effective in reducing the accumulation of leukocytes in the myocardium, preserving coronary blood flow, and attenuating the size of myocardial infarct during reperfusion [81,82]. Although E-selectin has been demonstrated to be upregulated in the reperfused myocardium , antibodies directed against E-selectin have not been effective in protecting the tissue against necrosis . Studies indicate that immunoneutralizing L-selectin on the membrane of neutrophils may reduce their accumulation in the myocardium. Administration of a L-selectin antibody to cats reduced myocardial necrosis by 50%, which coincided with an attenuation in neutrophil accumulation and coronary artery dysfunction . Given a similar cardioprotection with either P- or L-selectin mAbs in this model, suggest that a common adhesive interaction between P- and L-selectin may be inhibited. Furthermore, SLe(x) analogs, that block E- and P-selectin, and SLe(x) oligosaccharides reduce neutrophil accumulation, creatine kinase activity and myocardial necrosis as well as preserve myocardial blood flow [84,85], demonstrating that the selectin family of cell adhesion molecules plays a critical in the development of myocardial reperfusion injury. ICAM-1 is constitutively expressed on myocardial endothelium and is markedly enhanced following ischemia and reperfusion . These observations are complemented by studies demonstrating that ICAM-1 mRNA increases after 1 h of reperfusion, with cardiac myocytes expressing increases in ICAM-1 mRNA at 6 h after reperfusion . Numerous studies have investigated the role of ICAM-1 in myocardial reperfusion injury. These studies have demonstrated that ICAM-1 mAbs are effective in reducing myocardial necrosis by 50 – 70% in rabbits [88,89], cats  and non-human primates . In the cat, the reductions in myocardial necrosis coincided with a substantial reduction leukocyte accumulation within the ischemic zone of the myocardium . Since neutralization of ICAM-1 affords cardioprotection to the myocardium, it is not surprising that studies examining the role of CD11/ CD18-mediated leukocyte adhesion also revealed a significant reduction in myocardial dysfunction. In addition, a role for the CD11/CD18 adhesion molecule is supported by evidence of an increase in the expression of CD11b/CD18 on the surface of leukocytes from
patients with unstable angina, suggesting that ischemic periods in humans may initiate an inflammatory response that is characterized by leukocyte activation. Early studies from Simpson et al.  demonstrated that a CD11b mAb partially protected the canine myocardium from 90 min of ischemia and 6 h of reperfusion. Administration of the mAb 15 min prior to reperfusion caused a 46% reduction in myocardial necrosis. In addition, immunoneutralization of CD11a or CD18 reduced myocardial necrosis by 50 and 68%, respectively . These data suggest that blocking CD18-dependent adhesive interactions may have greater therapeutic effect compared to either CD11a or CD11b mAbs alone. In addition to reducing myocardial necrosis, the anti-CD18 mAbs were shown to reduce neutrophil accumulation in the myocardium and preserved endothelial cell-dependent vasorelaxation of coronary arteries . Anti-CD18 antibodies are also effective in restoring coronary blood flow and left ventricular developed pressure , however, antibodies to CD11a were less effective. Similar reductions in myocardial infarct have been observed in mice where immunoneutralization of either ICAM-1 or CD18 by gene-targeting resulted in a 60% reduction in the area of necrosis following reperfusion . In addition to an increase in the surface expression of leukocyte and endothelial cell adhesion molecules, substantial evidence exist describing changes in the circulating levels of cell adhesion molecules. Following acute myocardial infarction, serum levels of soluble E-selectin increase significantly 6–8 h after admission [95,96]. The rise in plasma E-selectin levels agree favorably with the kinetics of endothelial cell associated E-selectin which reaches peak levels at 3–4 h and declines thereafter, suggesting that the E-selectin is shed from the cell surface. Levels of soluble ICAM-1 were also observed to increase following acute myocardial infarction. In patients, ICAM-1 levels were elevated 6 h after admission, with significant levels remaining in the circulation up to 48 h . The elevations in soluble E-selectin and ICAM-1 were observed to positively correlate with leukocyte count and peak creatine kinase level, which represents the severity of myocardial infarction . This is supported by evidence demonstrating that patients experiencing fatal acute myocardial infarction had significantly greater levels of soluble E-selectin and ICAM-1, compared to patients who survived . Furthermore, patients undergoing thrombolysis displayed an invariance in soluble E-selectin and soluble ICAM-1, compared to patients not receiving treatment. Soluble P-selectin levels are also elevated after acute myocardial infarction for 1–3 days, suggesting the activation of platelets and endothelial cells . In other myocardial disorders such as unstable angina, significant levels of soluble E-selectin, P-selectin and ICAM-1 have also been observed . Soluble P-selectin increased within 1
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h of the development of angina, indicating a rapid release of P-selectin from platelets and/or endothelial cells . The role of these soluble adhesion molecules in mediating myocardial dysfunction remains unclear. However, data exists demonstrating that soluble adhesion molecules may inhibit leukocyte adhesion to endothelium [61,99] and thus, may limit tissue injury. Studies addressing the source and function of these soluble adhesion molecules, as well as their relationship to clinical outcome are warranted. Several inflammatory mediators including complement, ROMs, platelet activating factor (PAF) and TNF-a are associated with myocardial ischemia. A role for complement in mediating leukocyte induced injury in the myocardium was demonstrated by a reduction in leukocyte infiltration and tissue injury through the depletion of complement [74,75]. Complement may induce injury to the myocardium via several different mechanisms. Complement may exacerbate tissue injury by upregulating P-selectin on endothelial cells or inducing neutrophil chemotaxis . Recently, Buerke et al.  demonstrated that blocking the classical complement pathway preserves the myocardium from injury after 48 h of reperfusion. Inhibition of complement abolished reperfusion induced P-selectin and ICAM-1 expression and neutrophil infiltration. ROM production is also associated with myocardial ischemia and reperfusion as evidenced that administration of superoxide dismutase (SOD) and catalase prior to reperfusion reduces infarct size . However, these ROM scavengers had no effect on infarct size when administered after reperfusion. Other investigators have also shown that SOD is cardioprotective in myodcardial reperfusion injury [103,104]. Studies using isolated perfused heart indicate that ROM production occurs in the reperfused myocardium, and that superoxide anion is the primary species of ROMs produced in response to reperfusion [105,106]. Evidence also exists that PAF and TNF-a are also involved in mediating myocardial injury. Serum levels of PAF and TNF-a are increased significantly during reperfusion, with peak levels occurring at 15 and 30 min, respectively . Following 60 min of reperfusion, macrophage-derived TNF-a is also elevated. Administration of cloricromene, a TNF-a inhibitor, reduced serum TNF-a levels by 9-fold . Inhibition of TNF-a production and PAF activity was effective in significantly reducing the area of necrosis and the levels of creatine kinase and myeloperoxidase [107,108]. In addition, TNF-a neutralization also blunted the elevation in soluble E-selectin , suggesting that inhibiting TNF-a activity reduces endothelial cell activation. Given that TNF-a and PAF are potent agonist of leukocyte adhesion, it is reasonable to presume that the attenuation in tissue injury with TNFa and PAF inhibitors is due partly to a reduction in leukocyte-endothelial cell interactions.
In addition to increased production of these pro-inflammatory mediators during myocardial ischemia, vascular dysfunction may also occur due to the reduction in nitric oxide (NO) release from endothelial cells, immediately after reperfusion . NO has anti-adhesive properties as evidence of the ability of NO-donors to reduce P-selectin expression  and leukocyte adhesion [8,110]. Ma et al.  showed that NO mediates leukocyte-endothelial cell interactions and that a reduction in NO release by endothelial cells may lead to reperfusion injury. In addition, administration of NOdonating compounds reduces the accumulation of PMN in the myocardium following reperfusion [111,112] suggesting that NO may a potent inhibitor of leukocyte-endothelial cell adhesion.
5. Cerebral ischemia and reperfusion injury: stroke Normal neuronal function requires a steady delivery of nutrients which is maintained when cerebral blood flow is above a critical homeostatic level [113,114]. A reduction in blood flow, as observed during cerebral ischemia, to B 20–25 ml/100 g tissue/min produces a decrease in neuron viability and tissue integrity, thus a prompt restoration of blood flow is critical . However, like many other tissues, reperfusion of ischemic cerebral tissue may lead to the production of an array of inflammatory mediators, such as oxygen radicals , which promote leukocyte adhesion and tissue damage (Fig. 2). Many studies of cerebral vascular injury have used either permanent occlusion of blood vessels or temporary occlusion of blood vessels with subsequent reperfusion as a means to produce an inflammatory response. In the former model, investigators have examined cerebral ischemic injury in mice, rats and non-human primates following occlusion of the middle cerebral artery. In general, occlusion of the MCA causes a reduction in blood flow to 10–20% of baseline values . In the rat, P-selectin is upregulated within 30 min after permanent middle cerebral artery occlusion (MCAO, ). After returning to baseline values by 1 h of MCAO, P-selectin levels rise in a time dependent manner reaching maximal level of expression at 6 h and decline back to basal levels by 96 h. The biphasic expression of P-selectin occurs due to its initial translocation from endothelial cells while the second phase relies on the de novo synthesis of P-selectin. Given that in vivo  and in vitro  studies have demonstrated that cerebrovascular endothelial cells do not translocate P-selectin from the Weibal Palade bodies following stimulation with histamine, it is possible that the rapid upregulation of P-selectin observed in these animal models may be due to another inflammatory stimuli. In non-human primates (NHP), 3 h of MCAO induced a
M.J. Eppihimer / Pathophysiology 5 (1998) 167–184
Fig. 2. Mechanism proposed to explain the accumulation of leukocytes in cerebral tissue exposed to ischemia and reperfusion. Elevation in ROMs, which is potentiated by NO, and inflammatory mediators induces an elevation in leukocyte and endothelial cell adhesion molecules, and consequently, an increase in leukocyte adhesion and emigration and tissue injury.
significant increase in P-selectin expression but not ICAM-1 expression  In the rat, MCAO upregulates ICAM-1 mRNA levels between 1 and 3 h, with peak levels at 6–12 h and sustained amounts lasting up to a few days [121,135]. In contrast, exposure of human brain endothelial cells to 16 – 20 h of hypoxia was insufficient to elevate ICAM-1 mRNA, suggesting that other inflammatory cells which are present in vivo are needed to induce ICAM-1 expression . E-selectin expression also increases within 2 h after MCAO. In rats, MCAO-induced E-selectin was observed to reached peak levels at 12 h, thereafter, E-selectin expression declined back to baseline values by 96 h . The levels of protein coincide with peak levels of E-selectin mRNA occurring at 12 h and remaining elevated for up to 2 days . The upregulation of P-selectin, E-selectin and ICAM1 levels in ischemic cerebral tissue agrees favorably with the time-dependent nature of neutrophil accumulation. Neutrophils are observed to sequester in the ischemic
territory within 30 min of MCAO and reach peak values in the vasculature by 12 h . Neutrophil emigration from the vasculature begins 4–6 h after MCAO, with maximum accumulation of neutrophils in the ischemic zone at 24 h . In rats, MCAO induced moderate neutrophil infiltration by 24 h but achieved maximum levels by 72 h . In contrast, moderate infiltration of lymphocytes into the ischemic zone did occur until 72 h after MCAO, with peak levels at 7 days . Given that the infiltrating leukocytes expressed CD11a, it is likely that ICAM-1 mediated the recruitment of leukocytes into the injured region. While it seems reasonable to presume that neutralization of adhesion molecules would reduce ischemic injury, studies have shown that treatment with either a E-selectin oligopeptide  or an anti-ICAM-1 mAb  was ineffective in reducing infarct volume during permanent MCAO. However, both treatments were effective in reducing infarct size in transient MCAO followed by reperfusion, suggesting that different cellular mechanisms are involved.
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Because of interest in reperfusion strategies (i.e. tissue plasminogen activator, tPA) for acute ischemic stroke, an understanding of vascular and cellular contributions have evolved. A role for leukocyte involvement in the pathogenesis of cerebral injury following reperfusion is substantiated by evidence that treatment of rats with anti-neutrophil serum inhibited myeloperoxidase activity and tissue necrosis . Following 1 h of MCAO in the rat, P-selectin was observed to reach peak levels at 8 h after reperfusion, thereafter the expression of P-selectin declined back to baseline values by 3 days . In non-human primates (NHP), 3 h of MCAO induced a significant increase in P-selectin expression, and following reperfusion, P-selectin expression remained elevated up to 24 h . Discrepancies between these studies may be due to differences in the time of ischemia (1 vs. 3 h) or species (rat vs. NHP). Immunoneutralization of P-selectin through gene targeting has also demonstrated that P-selectin plays an important role in cerebral injury. In P-selectin deficient mice subjected to 45 min of MCAO, a significant reduction in the accumulation of neutrophils was observed 22 h after reperfusion, compared to wild-type mice . These studies demonstrated that P-selectin deficient mice had a significant reduction in cerebral infarct volume and an improved survival rate, compared to wild-type mice, suggesting that blocking P-selectin function may be a potential treatment for stroke. In non-human primates (NHP), maximum expression of E-selectin occurred after 3 h of MCAO and 24 h of reperfusion . An absence of E-selectin was observed in the contralateral non-ischemic tissue. After reperfusion, a significant elevation in E-selectin was observed in the contralateral tissue  suggesting a remote tissue injury response to the initial ischemic episode. However, the significance of E-selectin in the non-ischemic contralateral tissue remains unknown. Treatment of rats with an E-selectin oligopeptide, attenuates the size of the cerebral infarct following 24 h of reperfusion . In addition, analogs of Sle(x) were also effective in reducing infarct volume by 40% and leukocyte infiltration by 60% . These data suggest that E-selectin may facilitate leukocyte adhesion and ischemic injury following reperfusion. In vitro reoxygenation of human cerebral endothelial cells following 4 h of hypoxia, resulted in elevations in ICAM-1 mRNA levels by 4 h. Thereafter, ICAM-1 mRNA levels declined to baseline over the 24 h reoxygenation hours . In rats, ICAM-1 expression on endothelial cells increased after 2 h of reperfusion, with maximum levels occurring at 46 h and returning to baseline levels by 7 days . The expression of ICAM-1 on endothelial cells agrees favorably with the significant accumulation of ICAM-1 mRNA in the rat brain within 1 h of the onset of ischemia. Peak levels of ICAM-1 mRNA were attained at 10 h of reperfusion
and persisted over 7 days . Similarly ICAM-1 expression increased significantly in NHP after 3 h of MCAO with 1 h of reperfusion, but declined back to baseline values by 24 h . Finally, data from human patients demonstrate an increase in ICAM-1 in microvessels of infarcted tissue and to a lesser degree (but significant) in microvessels of non-infarcted tissue . These patients also exhibited an accumulation of neutrophils as early as 15 h after injury, peaked at 2 days and returned to normal levels by day 6. CD18 expression on leukocytes in patients with stroke also increased, compared to controls . Given the enhanced expression of ICAM-1 and CD18 in animals and patients with stroke, neutralization of these adhesion molecules seems to be an appropriate strategy to reduce leukocyte-mediated damage in stroke. Rats were administered mAbs against ICAM-1 and CD18 upon reperfusion after MCAO . These studies revealed that both mAbs caused a significant reduction in infarct volume and ischemic cell damage. Administration of anti-CD18 mAbs at 1 h after reperfusion also reduced cerebral lesion volume and leukocyte accumulation . Furthermore, neutralization of ICAM-1 by mAbs [122,140] and by gene-targeting  reduce cerebral infarct volume, supporting the premise that neutrophil adhesion in ischemic/reperfused tissues may be deleterious and blocking leukocyte adhesion to cerebrovascular endothelial adhesion reduces ischemic injury. Like other inflammatory responses, soluble isoforms of endothelial cell adhesion molecules have also been detected in the serum of patients with stroke. Evidence exists that soluble E- and P-selectin levels are elevated in patients with acute ischemic stroke [33,142]. Soluble E-selectin was observed to increased in a time-dependent manner after the onset of symptoms . Peak levels of soluble E-selectin occurred at 8 h and returned to baseline values by 3 days . Given that both selectins are released into the plasma, suggests that the source of the soluble selectins is endothelial cells. However, it can not be ruled out that a portion of the soluble P-selectin in the plasma is derived from platelets. Inconsistencies on the release of soluble ICAM-1 into the plasma exist in the literature. Early studies indicated that soluble ICAM-1 levels decline following acute ischemic stroke  while an increase in soluble ICAM-1 levels were observed in later studies . More recently, Frijns et al.  revealed an invariance of soluble ICAM-1 levels after acute ischemic stroke, compared to control conditions. The discrepancies may be accredited to differences in the underlying vascular risk factors of the subjects between studies. Although several cell types including endothelial cells and astrocytes express ICAM-1 , a major source of soluble ICAM-1 in the cerebral circulation is the endothelial cells . In light of these studies, soluble ICAM-1 has been demonstrated to mediate the
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adhesion of leukocytes to cerebrovascular endothelial cells . Pretreatment of activated leukocytes with recombinant soluble ICAM-1 reduced their adhesion to cerebral endothelial cells in a dose dependent manner. A similar inhibition of leukocyte adhesion was observed after leukocytes were incubated in serum containing soluble ICAM-1 . While the data strongly demonstrates that leukocyte and endothelial cell adhesion molecules are upregulated in response to either ischemia or reperfusion, the mechanism responsible for their induction remains unclear. Increases in the levels of IL-1b, TNF-a and IL-6 mRNAs have been observed in the ischemic brain [147– 150]. TNF-a mRNA increases 1 h after MCAO and reaches peak expression at 12 h before declining back to normal values by 5 days [149,151]. In contrast, IL-1b is induced much later, with elevations in mRNA at 6 h, maximum levels at 12 h, thereafter diminishing over 5 days . These inflammatory mediators upregulate the expression of endothelial cell adhesion molecules in a time-dependent manner  which parallels the kinetics of cytokine production in stroke, suggesting that cytokine production may be responsible for the leukocyte adhesion and cellular injury observed in cerebral ischemia. This is substantiated by studies demonstrating that neutralization of the IL-1 receptor, during MCAO, significantly attenuated the area of injury by 24 h of reperfusion . In addition to elevations in cytokine levels during cerebral ischemia, other inflammatory mediators such as ROMs, PAF and leukotriene B4 (LTB4) have been implicated in mediating ischemic injury. In rats, significant increases in LTB4 were observed during 30 min of ischemia and immediately after reperfusion . A 5-lipoxygenase-inhibitor and PAF antagonist reduced LTB4 concentrations after reperfusion but not during ischemia. Given that PAF and LTB4 are potent agonists of leukocyte adhesion and emigration, reducing tissue levels of these lipid mediators may attenuate the accumulation of leukocytes and, subsequently cell damage. This is supported by reductions in tissue injury by PAF antagonists in other models of ischemia and reperfusion injury . In an experimental model of stroke, tissue levels of PAF increased 20-fold by 25 min of ischemia and 2 h of reperfusion . Administration of a PAF antagonist during ischemia restored microvascular blood flow and attenuated edema formation. It is possible the PAF antagonist attenuated leukocyte adhesion and emigration, thereby reducing the level of vascular permeability in the reperfused region. Finally, ROMs have also been reported to be involved with the development of cerebral ischemia and reperfusion injury. Matsuo et al.  found that reperfusion enhances the production of ROMs and may be attenuated by neutrophil depletion. In addition, admin-
istration of a spin trapping agent significantly reduced cerebral infarct volume and improved mitochondrial function, following MCAO and reperfusion [157,158]. ROMs have also been demonstrated to induce ICAM-1 expression on cerebrovascular endothelial cells following reoxygenation , as evidence by an inhibition of ICAM-1 expression with N-acetyl-L-cysteine treatment. However, allopurinol had no effect in attenuating hypoxia- and reoxygenation-induced ICAM-1 expression, suggesting that the production of ROMS is not dependent on xanthine-oxidase (XO). Inconsistencies of the role of XO exist in the literature. Mink et al.  were unable to demonstrate the formation of XO in the injured brain whereas significant amounts of XO were reported in studies after 30 min of ischemia without reperfusion . A possible source of ROMs in cerebral ischemic injury may be the reaction of NO with superoxide anion to yield peroxynitrite, which decomposes to yield hydroxyl anions. NO concentration increases after induction of focal ischemia and declines after 1 h, but rises again during reperfusion . It has been suggested that the rise in NO production during reperfusion is due to the restoration of a supply of oxygen and L-arginine to the tissue . During MCAO with reperfusion, inducible nitric oxide synthase is elevated in macrophages/microglia and astrocytes . Treatment of animals with aminoguanidine, an iNOS inhibitor, reduces cerebral infarct volume when administered 24 h after reperfusion . In addition to iNOS, neuronal NOS (nNOS) is another an important source of NO under pathological conditions. Neutralization of nNOS by chemical inhibitors reduces infarct volume induced by transient MCAO . Furthermore, mice-deficient in nNOS are also protected from ischemic injury when subjected to transient MCAO , suggesting that NO may play an important role in mediating neurotoxicity in cerebral ischemia.
6. Atherosclerosis Although leukocyte-endothelial cell adhesion is generally associated with an occurrence in the post-capillary venules of the microvasculature, the expression of adhesion molecules has been demonstrated on the surface of endothelial cells overlying atherosclerotic lesions in large conduit arteries. It has been proposed that the fatty streak lesions observed in atherosclerosis arise from an accumulation of leukocytes in the vessel intima, a differentiation of monocytes into macrophages, and their subsequent uptake of cholesterol esters that give rise to foam cells [165–168]. Several leukocyte subsets are associated with atherosclerosis including neutrophils, lymphocytes and monocytes; however, monocytes have received a large amount of attention
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due to their differentiation into macrophages . In addition, emigrated leukocytes may release a variety hydrolytic enzymes and ROMs that may induce injury to neighboring cells and initiate the proliferation of atherosclerotic lesions (Fig. 3). An elevation in low density lipoproteins is generally associated with the progression of atherosclerosis in humans and animals . Exposure of endothelial cells to LDL increases their adhesiveness to leukocytes [171,172], and oxidation of the LDL markedly increases the pro-adhesive effect of LDL [173,174]. Oxidized LDLs induce P-selectin expression on endothelial cells within 1 hwith a persistent expression of P-selectin up to 4 h before declining back to baseline values . Although this expression resembles that of ROM induced-P-selectin expression , it can not be ruled out
that oxidized LDLs induce P-selectin through a transcriptional-dependent mechanism. An elevation in endothelial cell adhesion molecules agrees favorably with observations that oxidized LDLs elevate the rolling and adhesion of neutrophils to arteriolar and venular endothelium . Similar adhesive interactions, in addition to increased vascular permeability, were observed in the rat mesentery . Antibodies directed against ICAM-1, CD18 and P- and L-selectin attenuated oxLDL-induced leukocyte adhesion and emigration, and vascular permeability . These studies demonstrated that oxidized LDL-induced leukocyte-endothelial cell adhesion plays an important role in disrupting the endothelial cell barrier. Furthermore, LDL receptor deficient mice that were fed a high fat diet exhibit significantly greater leukocyte adhesion and emigration
Fig. 3. Mechanism proposed to explain the accumulation of leukocytes in atherosclerotic lesions. Elevation in ox-LDLs produces a concomitant reduction in NO. This is paralleled by an increase in ROMs and other inflammatory mediators. A subsequent increase in leukocyte and endothelial cell adhesion molecules leads to an increase in leukocyte adhesion and emigration. The release of inflammatory mediators from emigrated leukocytes produces a chemotactic signal, which amplifies the magnitude of leukocyte adhesion and emigration. The differentiation of monocytes into macrophages, and their uptake of cholesterol produces the formation of foam cells. In addition to foam cells, growth factors released by emigrated leukocytes induce cell proliferation and consequently, the development of an atherosclerotic lesion.
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under basal and inflammatory conditions, compared to LDL receptor mice and wild type mice on a normal diet . LDL receptor deficient mice that were fed a high fat diet also had a greater plasma level of TNF-a, following endotoxin administration. The exacerbation of the inflammatory response may be due to the presence of oxidized LDL in these mice. Infusion of oxidized LDL into animals exposed to ischemia and reperfusion injury produced a significant increase in leukocyte adhesion and emigration but not vascular permeability, indicating the ox-LDL acts synergistically with ischemia and reperfusion to promote leukocyte adhesion and emigration . These studies also revealed that oxidized LDLs did not elicit an enhanced inflammatory response during hypertension. Given that oxidized LDL may directly inactivate NO and inhibit its formation and release, the elevated leukocyte adhesion may be contributed to a deficiency in NO . Liao and Granger  showed that treating animals with a NO-donor attenuated oxidized LDL-induced leukocyte adhesion and emigration and vascular permeability, suggesting that NO protected the vasculature against the deleterious effects of oxidized LDLs. Furthermore, studies in hypercholesterolemic animals have demonstrated that administration of the NO-precursor, L-arginine, enhances the release of endothelium derived NO, reduces monocyte adhesion, and reduces the area of preexisting atherosclerotic lesions [180–182]. In humans with hypercholesterolemia, dietary supplementation with L-arginine reduces the adhesiveness of mononuclear leukocytes to endothelial cells . A similar effect of a NO-donor on monocyte adhesiveness was also found in this study. Exposure of monocytes from patients with hypercholesterolemia to sodium nitroprusside significantly attenuated their adhesion to endothelial cell whereas sodium nitroprusside had no effect on the adhesiveness of monocytes derived from normal patients . However, the inhibitory effect of NO on leukocyte adhesion in the progression of atherosclerotic lesions in human remains unknown. Since leukocyte adhesion is regulated in part by the expression of endothelial cell adhesion molecules, numerous studies have examined the ECAM levels in atherosclerotic plaques. In humans and experimental animals, an increase expression of P-selectin , E-selectin [185,186], ICAM-1 [184,187] and vascular cell adhesion molecule-1 (VCAM-1)  were observed on endothelium overlying atherosclerotic plaques; however, the evidence for VCAM-1 is conflicting in humans [186,189]. P-selectin is coexpressed with ICAM-1 expression on the endothelium , suggesting that these molecules may act synergistically to mediate leukocyte recruitment. These findings agree with observations from gene-targeted mice that were fed a high-cholesterol diet. In ICAM-1/P-selectin deficient mice, a 71% reduction in atherosclerotic lesion area was observed,
compared to wild-type mice . In addition, mice deficient in either P-selectin, ICAM-1 or CD18 exhibited significant reductions in lesion area. Mice deficient in both ICAM-1 and CD18 demonstrated the greatest reduction in lesion area with a value of 76%. The decrease of fatty streaks in gene-targeted mice may be attributed to a reduction in the recruitment of monocytes into the vessel wall since monocytes express ligands for P-selectin, ICAM-1, and CD18. In rats experiencing hypercholesterolemia, ICAM-1 expression was significantly increased on the endothelial cells of the aorta, compared to control rats . This exacerbation in ICAM-1 expression coincided with an increase in the accumulation of macrophages and T lymphocytes in the lesion prone area. Administration of mAbs directed against ICAM-1 and LFA-1 were effective in reducing macrophage but not T-lymphocyte adherence and emigration into the intima . These data indicate that the ICAM-1/LFA-1 adhesion pathway is involved during the early stages of cholesterol induced atherosclerosis. In previous reports, soluble levels of endothelial cell adhesion molecules have been demonstrated to increase in patients with atherosclerosis, compared to control subjects [142,192,193]. In carotid atherosclerosis, soluble P-selectin and E-selectin levels increase approximately 40% compared to controls . In this patient group, soluble VCAM-1 and ICAM-1 was invariant between groups. In a larger patient population, E-selectin and ICAM-1 but not VCAM-1 levels, were significantly elevated in atherosclerotic patients . In contrast, soluble VCAM-1 but not soluble E-selectin, P-selectin, or ICAM-1 levels were significantly elevated in patients with atherosclerosis . In this study, the level of circulating VCAM-1 was highly correlated to the atherosclerotic area. Furthermore, Nakai and coworkers  showed that elevations in VCAM-1 mRNA expression coincided with increases in soluble VCAM-1 levels in patients with aortic atherosclerosis. These findings suggest that the level of soluble VCAM1 may be an appropriate indicator for the amount of VCAM-1 expressed in an atherosclerotic lesion. Inconsistencies between these studies may be due to the location of the atherosclerotic lesion (carotid vs. aortic) or other vascular risk factors, but it remains unclear the role of these soluble adhesion molecules in the development of atherosclerosis.
7. Summary Much progress has been made in delineating the role of leukocyte-endothelial cell adhesion in the pathogenesis of cardiovascular disease. The cell adhesion molecules and inflammatory mediators that are responsible for mediating leukocyte trafficking in these disease
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states have been established. This information has yielded new therapeutic strategies that may be useful in the treatment of acute and chronic cardiovascular pathologies, however future advancements in the management of these diseases will probably come from altering cellular responses at the genetic level. These studies will presumably utilize the growing number of genetically engineered animals to examine the progression of cardiovascular diseases. In addition, future work in neutralizing leukocyte-endothelial adhesion will require that therapeutic agents be site specific, that is, be able to target specific organs. This will enable physicians to treat cardiovascular diseases while maintaining a non-compromised immune system.
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