Leukotrienes in Cardiovascular Diseases

GIANCARLO FOLCO, GIUSEPPE ROSSONI, CAROLA BUCCELLATI, FERRUCCIO BERTI, JACQUES MACLOUF, and ANGELO SALA

Center for Cardiopulmonary Pharmacology, Institute of Pharmacological Sciences, University of Milan, Milan, Italy

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

One surprising feature that is clear from even a superficial overview of the current literature on leukotrienes (LTs) and antileukotriene drugs is represented by the fact that biomedical research in this field has been markedly polarized on asthma and allergic disorders and has largely overlooked other diseases that are also based on the existence of an inflammatory process with increased vascular permeability, edema, and the presence of a heterogeneous cellular infiltrate.

In other diseases, in particular those related to the cardiovascular system, our current understanding of the role of LTs, although far from complete, seems to indicate enough potential for an involvement of LTs in cardiac, hemodynamic, and microcirculatory pathophysiology and may therefore justify antileukotriene therapy. Sulfidopeptide-LTs do possess a unique pharmacological profile, characterized by potent constrictor actions on the microvasculature; they can enhance permeability, reduce coronary blood flow, and reduce myocardial contractility and cardiac output without affecting the heart rate (1). These actions "per se" could provide an unexpected key to interpret pathologic processes (unstable angina?) and could lead to alternative means of treating certain diseases in future. Moreover, LTs can be formed from arachidonic acid by perivascular mast cells (5), but can also be synthesized by platelets and, probably more importantly, by endothelial and vascular smooth muscle cells, from neutrophil-derived LTA₄. This process of cell cooperation is likely to be accelerated by, e.g., ischemia-reperfusion and lead to the occurrence of a high local bioavailability of cysteinyl-leukotrienes (Cys-LTs) within the endothelial cell, as a likely direct consequence of leukocyte adhesion, leading to its retraction, and to a facilitated emigration of polymorphonuclear leukocytes (PMNs) from the circulating pool. This may add a new dimension to the traditional view of the cardiovascular complications that accompany an inflammatory response induced by leukocytes in myocardial tissue, which could be driven by Cys-LTs more than by their endogenous chemotactic metabolite LTB₄. A higher than normal PMN count is indeed considered a predisposing factor to cardiac infarction and increased urinary excretion of LTE₄ has been reported (6) after episodes of unstable angina and acute myocardial infarction. Moreover, endothelial cells do possess a unique isoform of glutathione S-transferase, GSTII (7), which catalyzes LTC₄ production in endothelial cells; this enzyme is different from LTC₄ synthase in platelets and may be the key enzyme in the transcellular biosynthesis of LTC₄ that takes place during PMN-endothelial cells interactions.

LTs, therefore, do respect the majority of Dale's criteria, which must be fulfilled in order to be considered important mediators of the inflammatory events that may ultimately lead to myocardial injury due, e.g., to regional ischemia and reperfusion. The majority of data accumulated thus far, indicating that cys-LTs are potent modulators of vascular tone, cardiac functions, and the microcirculation, has been obtained with the use of exogenous, pure synthetic LTs and may therefore mimic only partially the effects of their generation in situ. We report here that the production of LTs from cellular sources may profoundly affect coronary function, and that selective lipoxygenase inhibitors provide significant cardioprotection.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hearts from male albino rabbits were isolated and perfused retrogradely in a recirculating system (50 ml), as previously described (8); coronary perfusion pressure (CPP) and left ventricular end-diastolic pressure (LVEDP) were monitored continuously. Rabbit hearts were perfused with purified human neutrophils (PMNs, 5 × 10⁶ cells) and challenged with A23187 (0.5 µM). At the end of the experiment, the entire heart reservoir fluid was withdrawn for storage under argon atmosphere at 20° C until high-performance liquid chromatography (HPLC) analysis (8).

Positive identification of Cys-LTs was obtained by UV spectral analysis of chromatographic peaks eluting at characteristic retention times. Quantitation was performed on positively identified peaks only, using standard curves of synthetic LTC₄/D₄/E₄ and LTB₄ (Cascade Biochemicals, Reading, UK) and values of Cys-LTs were corrected for recovery.

In Vivo Experiments

Male albino New Zealand rabbits were anesthetized with pentothal sodium (30 mg/kg) and the left side of the thorax was opened through the fourth-fifth intercostal space, to permit free access to the left ventricular myocardium. The left anterior descending coronary artery was separated from the myocardial tissue and ligated with a silk 6.0 suture, armed with an atraumatic needle. A 72-h recovery period was chosen according to the original report (9), in order to fully appreciate the mortality rate.

Rabbits were randomly divided in three experimental groups: sham- operation (SH), permanent coronary artery ligature (CAL), and CAL in rabbits treated with BAY X1005 (CAL + BAY X1005). Treatment was performed according to the following protocol: SH and CAL groups were infused with the solvent (absolute ethanol, 33 µl/min/kg), whereas the CAL + BAY X1005 group was infused with BAY X1005 (dissolved in absolute ethanol) at a dose of 10 mg/kg/h for 2 h. Rabbits were infused at a flow rate of 0.1 ml/min starting immediately after anesthesia and lasting for 2 h. The second hour of infusion started at the beginning of the surgical procedure.

Electrocardiographic Recording

The electrocardiogram (ECG) was performed according to the conventional method of Einthoven, setting the electrodes in lead II and connecting them to the recording apparatus (Cardioline Delta 1; Remco Italia, Milan, Italy).

Rabbits were subjected to the first ECG recording immediately after anesthesia and 0.5 and 72 h after coronary artery ligation.

Myeloperoxidase Assay in Rabbit Myocardium

The hearts from 72-h surviving rabbits were used for determination of tissue myeloperoxidase (MPO) activity, assayed by measuring the H₂O₂- dependent oxidation of 3,3', 5,5'-tetramethylbenzidine (TMB), using a 96-well microtiter plate-modified microassay (10).

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In Situ Formation of Cys-LTs and Coronary Function: Modulation by Nitric Oxide

The Isolated rabbit hearts, perfused at a constant flow of 20 ml min¹, for 30 min, demonstrated an average CPP of 45 ± 1.4 mm Hg; resistance to perfusion pressure did not change when unstimulated human PMNs (5 × 10⁶ cells) were perfused through the isolated rabbit heart. However, challenge with the calcium ionophore A23187 (0.5 µM), in the presence of PMNs, brought about a significant increase in CPP that started approximately 10 min after challenge and continued thereafter (134 ± 4 mm Hg, at 30 min). Prechallenge LVEDP values were 5 ± 0.1 mm Hg and increased to 11.7 ± 6.7 mm Hg after challenge with A23187 in the presence of PMNs. Pretreatment (30 min before challenge) of the rabbit hearts with L-arginine (100 µM) did not affect coronary tone but resulted in a significant protection against its PMN-dependent increase.

Pretreatment (20 min before challenge) of rabbit hearts with N^G-monomethyl-L-arginine (L-NMMA, 10 µM) caused a progressive increase in CPP that was slow in onset and unaffected by A23187 (0.5 µM) (Figure 1) (11). When L-NMMA-treated hearts were challenged in the presence of PMNs, a marked increase in CPP was seen, which was fast in onset and so pronounced as to cause cardiac arrest in systole in five of six hearts at 20 min (Figure 1). LVEDP as well increased markedly to 47 ± 16 mm Hg (p < 0.01 versus control PMNs).

View larger version (17K): [in this window] [in a new window]   Figure 1.   Effect of pretreatment (20 min) with L-NMMA (10 µM) on coronary perfusion pressure (CPP; left) and levels of sulfidopeptide leukotrienes (Cys-LTs; right) of rabbit isolated hearts perfused under recirculating conditions, with human PMNs (1 × 105 cells ml1) and challenged with A23187 (0.5 µM) (open triangles; n = 6), or challenged with A23187 in the absence of PMNs (open diamonds; n = 3), or challenged with vehicle (ethanol) (solid diamonds; n = 3). Cys-LTs were analyzed by reversed-phase HPLC after extraction of whole buffer reservoir fluid, withdrawn 30 min after challenge. Values represent means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 versus PMN-perfused heart. [Reprinted by permission from Buccellati, C., and coworkers (Reference 11).]

The striking increase in CPP observed in the presence of L-NMMA was fully prevented by L-arginine (120 ± 5 mm Hg, at 30 min). L-Arginine pretreatment was also able to prevent the increase in LVEDP at 30 min after A23187 challenge of recirculating PMNs; in fact, LVEDP values observed under these conditions were not different from prechallenge values (6 ± 1 mm Hg, p < 0.01 versus L-NMMA).

Leukotriene Production of the PMN-perfused Isolated Heart of Rabbit

HPLC analysis of the final volume of the recirculating perfusate showed that, in the absence of PMNs, Cys-LTs (LTC₄ and LTD₄) were almost undetectable. However, when challenge with A23187 was carried out in the presence of PMNs, formation of Cys-LTs took place and allowed their positive identification by on-line UV spectrum analysis; after pretreatment with L-arginine, the amount of Cys-LTs detected in the perfusion buffer was significantly reduced (Figure 1).

Pretreatment of the rabbit hearts with L-NMMA, followed by challenge with A23187 in the presence of PMNs, led to a marked increase in Cys-LT formation compared with untreated hearts (Figure 1), which was fully reversed by L-arginine.

Effect of BAY X1005 on Rabbit Mortality Rate and Creatine Phosphokinase Activity

The ligation of the anterior coronary artery in rabbits resulted in acute myocardial infarction of the left ventricular wall, marked by a mortality rate of 65% at 72 h as compared with sham-operated animals (p < 0.001) (Figure 2) (12). In contrast, intravenous treatment of rabbits with BAY X1005 (10 mg/kg/h for 2 h) resulted in clear myocardial protection, with a remarkable reduction of the mortality rate, which, at 72 h, was 25% compared with sham-operated rabbits (NS).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Cardioprotective effects of BAY X1005 in the rabbit after permanent coronary artery ligation. [Reprinted by permission from Rossoni, G., and coworkers (Reference 12).]

The results obtained from the analysis of plasma creatine phosphokinase (CPK) activity were in line with the preceding findings. In fact, permanent ligature of the coronary artery of rabbits caused a significant increase in CPK activity at 72 h (903 ± 22 U/L, n = 6, p < 0.001 versus SH) as compared with the sham-operated animals (495 ± 12 U/L, n = 10), whereas in the group of BAY X1005-treated animals, the activity of this enzyme (543 ± 27 U/L, n = 8) was not significantly different from that measured in controls (SH).

Effect of BAY X1005 on Myeloperoxidase Activity in Rabbit Myocardium

Experimental studies have shown PMN margination in venules at the periphery of an infarct after permanent coronary occlusion and MPO activity is considered to be a reliable index of tissue neutrophil infiltration. The results obtained in these experiments clearly demonstrate that, 72 h after coronary artery ligation, the MPO activity measured in the heart tissue corresponding to the central ischemic area of the ligature on the left anterior descending coronary artery of the surviving rabbits was increased twofold (11.3 ± 2.7 IU/g tissue) when compared with sham-operated animals (5.1 ± 1.3 IU/g tissue, p < 0.05). However, in the rabbits treated with BAY X1005 (10 mg/kg/h for 2 h) the level of myocardial tissue MPO activity was in the range of that measured in control (SH) animals (5.0 ± 1.3 IU/g tissue).

Effect of BAY X1005 on the Electrocardiographic Pattern

In these experiments the electrocardiogram (ECG) was recorded in lead II. The ECG tracings obtained before coronary artery ligation were comparable for heart rate values and for the absence of significant abnormalities of depolarization and repolarization pattern. The analysis of lead II ECG obtained from sham-operated rabbits 0.5 and 72 h after surgery did not show ST segment or T wave changes; cardiac arrhythmias were totally absent. Only the QRS complex was slightly reduced and in some cases a right-axis deviation was present. These electrocardiographic features could be attributed to the effect of anesthesia and to the surgery procedure (pericardium section).

ECG recordings, monitored ~ 0.5 h after coronary artery ligation (CAL), were characterized by up- and downward shifts of the ST segment in both groups of rabbits (CAL and BAY X1005 treated); these alterations were distinctly more severe in the CAL group of rabbits.

The evaluation of the electrocardiograms of the surviving rabbits 72 h after coronary artery ligature showed the presence of a deep Q wave or of a decrease in amplitude of the initial R wave, associated with persistent ST segment elevation, suggesting that an extensive transmural myocardial infarction had occurred. These ECG abnormalities were often accompanied by heart rate slowing and ventricular arrhythmias. The infusion of BAY X1005 (10 mg/kg/h for 2 h) to the rabbits brought about a clear protection against ECG derangement induced by coronary artery ligation. In fact, none of the records obtained at 72 h showed alterations of the ST segment or cardiac rhythm. These ECG tracings were not different from those recorded before coronary artery ligature and are comparable to those obtained in sham-operated rabbits.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The cooperation between PMNs and coronary endothelial cells results in significant in situ transcellular biosynthesis of sulfidopeptide-LTs and altered coronary tone. The formation of Cys-LTs from cellular sources causes profound vasoconstriction and perivascular edema, resulting in severe derangement of hemodynamic and cardiac functions both in vitro and in vivo. These findings extend previous studies (13) since the effects observed could be reproduced only partially by simple addition of high amounts (2 µg) of exogenous LTC₄ in bolus (our unpublished observations, 1999), pointing to the primary importance of local concentrations of Cys-LTs acting on target cells at the site of production, in good agreement with their autacoid nature. In this respect PMNs are cells that seem to be intended for transcellular biosynthesis of Cys-LTs, since a substantial fraction of their primary arachidonic acid (AA) metabolite, i.e., the unstable epoxide leukotriene A₄ (LTA₄), is released outside the cells, where it becomes available to neighboring acceptor cells (e.g., endothelial cells, ECs) for further metabolism (14). This could lead to high local bioavailability of Cys-LTs, which are known to constrict coronary arteries (15) and have vasopermeant properties (16) by contributing to endothelial cell retraction phenomena. This might have relevance for the extravasation of white cells from the vessel lumen to the tissue. The interaction between PMNs and ECs is controlled by a variety of factors that regulate the trafficking and migration of leukocytes. Both cells express on their surface proadhesive molecules, with diverse structures and mechanisms of expression, which may act as receptors to tether the two cells together or as signals that induce activation-dependent adhesion events (17). Less is known about locally produced factors that exert an inhibitory influence on leukocyte adhesion.

Nitric oxide has attracted considerable interest as part of a widespread intercellular communication system. Continuous generation of NO has been shown to play an important role in setting the resting tone of systemic as well as coronary resistance vessels (18); in addition, NO exerts several other effects including inhibition of PMN activation and adherence (19, 20). These properties may be of considerable importance in explaining the modulatory effect of NO on the transcellu- lar biosynthesis of Cys-LTs, taking place between PMNs and coronary endothelial cells; moreover, they indicate that the cardioprotective effect of NO may not be simply restricted to the control of the coronary vascular tone, but may reside in its capacity to modulate the cross talk between circulating cells and vessel wall (21).

The biological role of NO in controlling vascular homeostasis may be potentially relevant in the pathogenesis of vascular diseases such as human coronary atherosclerosis, where the capacity of the endothelium to synthesize NO is reduced, or cardiac infarction and ischemia reperfusion injury, where PMNs are active participants in propagating tissue damage and increased urinary leukotriene excretion has been reported (22). Research directed at the prophylactic use of agents that are able to prevent the adhesion of PMNs to the vascular endothelium and to cut off the biochemical interplay between these two cell populations, may prove effective in reducing the incidence of ischemic heart disease and myocardial infarction. In this respect, the most intriguing findings obtained in our experiments were related to the electrocardiographic pattern (normal lead II) of the surviving animals, showing that the severe abnormalities of the ECG tracings observed at 72 h after coronary artery ligation, i.e., ST segment elevation, Q wave depression, and cardiac arrythmias, were abolished after selective 5-lipoxygenase (5-LO) inhibition with the 5-LO-activating protein (FLAP) inhibitor, compound BAY X1005. In addition, the significant reduction in overall mortality rate as well as the life-saving effect of BAY X1005 during the first 24 h postligature, and the normalization of the MPO content of the affected cardiac area subjected to acute coronary artery ligation, are quite promising and suggest that the potential therapeutic value of leukotriene inhibitors needs to be evaluated. However, the available data concerning the use of antileukotriene drugs provide a still rather heterogeneous and controversial picture. 5-LO inhibitors as well as a few Cys-LT and LTB₄ receptor antagonists have been tested in models of experimental myocardial infarct, with or without reperfusion, in different animal species. Promising results have been obtained with the use of selective 5-LO (or FLAP) inhibitors or with dual LO-cyclooxygenase (COX) blocking agents (23) with reduction of infarct size. Interestingly enough, the most favorable results have been reported with the use of compound REV-5901 (27), which shows a combined activity as inhibitor and antagonist of LTs. However, failure to reduce the extent of myocardial infarct size after occlusion-reperfusion in the dog has also been reported, in spite of persistent inhibition (~ 80%) of LTB₄ production in blood challenged ex vivo with zymosan (28).

Much more controversial is the picture that emerges from studies with LT receptor antagonists; indeed, findings suggesting a role for Cys-LTs in the extension of ischemic damage and in cardiac dysfunction during reperfusion (29) are evenly balanced by results suggesting that these autacoids do not contribute to the progression of myocardial ischemic/reperfusion injury (32).

It is not easy to find a comprehensive explanation for these apparently contradictory results; LT synthesis inhibitors could provide a more effective salvage of ischemic myocardium by preventing formation of vasoactive LTs (Cys-LTs) as well as chemotactic LTs (LTB₄). On the other hand, the LT receptor antagonists investigated so far may not display enough potency to compete with the endogenous ligands (Cys-LTs, LTB₄) that are likely to be present with high local bioavailability at sites where inflammatory cell accumulation and myocardial injury take place.

Although preclinical and clinical evidence (35) indicates that the 5-LO pathway becomes activated in coronary artery disease and suggests that LTs may play a pathogenic role, we are still missing the results of properly planned and controlled, phase II clinical trials with the use of anti-LTs. With a few recent exceptions (36), this is also true for conditions such as rheumatoid arthritis, psoriasis, and glomerulonephritis.

In conclusion, the demonstration that the production of LTs from cellular sources alters coronary function suggests that they may play a role in coronary heart afflictions. The significant cardioprotection provided by selective lipoxygenase inhibitors gives emphasis to LTs as potential targets in cardiovascular disease and may provide the necessary background and justification to launch novel therapeutic programs.

	Footnotes

Correspondence and requests for reprints should be addressed to Giancarlo Folco, Ph.D., Center for Cardiopulmonary Pharmacology, Institute of Pharmacological Sciences, University of Milan, 20133 Milan, Italy. E-mail: giancarlo.folco{at}unimi.it

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