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Ann Thorac Surg 1999;68:442-446
© 1999 The Society of Thoracic Surgeons

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Original Articles

Influence of polymorphonuclear leukocytes and plasma on coronary vasomotion after ischemia

^a Medizinische Klinik und Poliklinik I, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin, Germany

Address reprint requests to Dr Felix, Medizinische Klinik und Poliklinik I, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Schumannstr 20-22, D-10098 Berlin, Germany
e-mail: stephan.felix{at}charite.de

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. The role of plasma and neutrophils in endothelial dysfunction following myocardial ischemia was investigated.

Methods. Isolated rabbit hearts were perfused at constant pressure with a modified Krebs-Henseleit solution. After a 30-minute global ischemia the hearts were perfused at the onset of reperfusion for 10 minutes with either NaCl (group I, n = 6), autologous plasma alone (group II, n = 5), autologous polymorphonuclear leukocytes alone (PMN, group III, n = 6), or PMN and plasma in combination (group IV, n = 5). Before and after ischemia the effects of intracoronary endothelial dependent and independent vasodilation by acetylcholine (1 x 10^-7mol/L) and glycerol trinitrate (1 x 10^-6mol/L) were investigated.

Results. A similar increase in coronary flow was induced in groups I, II, and III by acetylcholine and glycerol trinitrate before and after ischemia. In contrast, in group IV the endothelial dependent vasodilation was significantly depressed (p< 0.05). In groups II and IV a moderate but significant reduction in the recovery of the left ventricular pressure was observed after ischemia and reperfusion.

Conclusions. These results suggest that after myocardial ischemia, plasma is required for neutrophil-mediated endothelial dysfunction.

	Introduction

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Reperfusion of the ischemic myocardium causes endothelial dysfunction [1, 2], expressed as impaired responses to endothelium dependent stimulators of nitric oxide (NO) release. Activation of polymorphonuclear neutrophils (PMN) is an important mechanism of reperfusion-induced endothelial dysfunction mediated primarily by oxygen-derived free radicals [3, 4]. Superoxide anions inactivate NO and initiate lipid peroxidation, thus altering membrane permeability and leading to endothelial cellular dysfunction [5]. Activated PMNs elicit an endothelium dependent vasoconstriction in rabbit aorta and cat coronary arteries that is caused by oxygen-free radicals-mediated inactivation of basal NO release [6, 7]. On the other hand, a decreased release of NO due to endothelial dysfunction induces an upregulation of cell adhesion molecules that further enhances the interaction of leukocytes with the coronary endothelium [8, 9]. An important first step in neutrophil adhesion and accumulation is the local generation of chemoattractants responsible for leukocyte recruitment [10]. Particularly, there is compelling evidence that plasma factors, most likely activation of the complement cascade, play a role in myocardial ischemia and reperfusion injury [11, 12]. In addition, complement activation directly attenuates endothelial dependent relaxation through the formation of the terminal attack complex [13]. However, it is presently unknown what role plasma factors play in the profound impairment of endothelial function that occurs after reperfusion of ischemic myocardium. Therefore the purpose of this study was to determine the role of plasma and PMNs in endothelial dysfunction during reperfusion after ischemia.

	Material and methods

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Isolated perfused heart preparation
Rabbits (New Zealand White, Germany) of either gender (1400–2000 g) were heparinized with 1000 U/kg and anesthetized with pentobarbital sodium (50 mg/kg iv). The hearts were rapidly excised and perfused according to the Langendorff technique at constant pressure (80 cmH₂O) with a modified Krebs-Henseleit solution containing (mmol/L): NaCl 118, KCl 4, MgSO₄ 1.2, KH₂PO₄ 1.19, NaHCO₃ 24.9, CaCl₂ 2.5, glucose 5, and pyruvate 2.0. The perfusate was equilibrated with 95% O₂–5% CO₂, 37°C, PH 7.4. Temperature was kept constant at 37°C during the whole ischemia and reperfusion period. Two side arms in the perfusion line located just proximal to the heart cannula allowed infusion of PMNs and plasma directly into the heart. Left and right ventricular pressure was recorded via a fluid-filled latex balloon inserted through the mitral valve and attached to a pressure transducer and chart recorder. The left ventricular end diastolic pressure was maintained at 5 mm Hg. Balloon pressure was electronically differentiated to yield dP/dt_max and heart rate. Coronary flow rates were monitored with an ultrasonic flow meter (Transsonic, Fuerstenfeldbruck, Germany) connected to a flow probe that was built into the aortic arch. Coronary perfusion pressure was monitored with a pressure transducer attached to the aortic perfusion cannula. All parameters were continuously displayed. Continuous registration included heart rate, left ventricular pressure (LVP), left ventricular peak positive dP/dt (LVdP/dt_max), coronary flow rates (CF), and coronary perfusion pressure (CPP).

The investigation conforms with the "Guide for Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication No. 85-23, revised 1985).

Isolation of rabbit PMNs
PMN were isolated from whole blood obtained from New Zealand White rabbits (1.4–2.2 kg) as previously described [14]. The blood was drawn into sodium citrate (3.15% sodium citrate, ratio 1:5) and mixed with dextran (500.000 mol wt, 6% in saline, ratio 1:5) and allowed to sit for 1.5 hours at room temperature for sedimentation of erythrocytes. The supernatant was centrifuged (500 g, 5 minutes) and the pellet was subjected to hypotonic lysis to eliminate contaminating erythrocytes. The cells were layered on Ficoll Hypaque (ratio 1:2) and then centrifuged once more for 20 minutes at 500 g. The final pellet was resuspended in calcium-free phosphate buffer saline (PBS) to a concentration of 1 x 10⁷ cells/mL. Cell viability, assessed by trypan blue exclusion, was > 99%.

Plasma—isolation
Rabbit autologous plasma was obtained by puncture of the thoracic aorta with a 10-mL syringe (+ EDTA). The sample was immediately centrifuged at 500 g, 5 minutes at 0°C. The upper plasma layer was subsequently filtered to a 0.2 µm micropore filter and stored in ice water until it was used in the isolated perfused heart.

Experimental protocol
The experimental protocol of ischemia/reperfusion was performed according to Shandelya and associates [15] (Fig 1). However, in the present study autologous plasma and PMNs were used. The rabbit hearts were perfused in the Langendorff mode. After a stabilization period at constant pressure–perfusion (80 cmH₂O)–global ischemia was initiated. Flow of Krebs’ perfusate was stopped, creating global zero-flow ischemia for 30 minutes. Four different groups were compared depending on the requisite media infused during the first 10 minutes of the reperfusion period:

Group I (control group, n = 6): Hearts were reperfused with NaCl at 2% of coronary flow.

Group II (n = 5): Autologous plasma and NaCl were infused into the aortic arch just above the coronary arteries at 1% of coronary flow, respectively, during the first 10 minutes of reperfusion.

Group III (n = 6): Autologous PMNs and NaCl were administered at 1% of coronary flow, each. The final concentration of PMNs was 1 x 10⁵/mL.

Group IV (n = 5): Hearts were reperfused with both plasma and PMNs (each 1% of coronary flow, final concentrations of PMNs 1 x 10⁵/mL).



Then, after 10 minutes, perfusion was continued with Krebs-Henseleit-solution alone for an additional 40 minutes, during which continuous registration of heart rate, LVP, left LVdP/dt_max, coronary flow rates, and coronary perfusion pressure was performed.

View larger version (28K): [in this window] [in a new window]   Fig 1. Schematic diagram of the experimental protocols. In group I (control group), hearts were infused with NaCl (0.9%) during the first 10 minutes of reperfusion after 30 minutes of global stop flow ischemia. In groups II, III, and IV, autologous polymorphonuclear neutrophils (PMN) alone, plasma alone, and both PMN and plasma in combination, were respectively infused intracoronarily during the first 10 minutes of reperfusion. Before and after global ischemia, responsiveness to the endothelial dependent and independent vasodilators acetylcholine (ACH 1 x 10-7 mol/L) and glycerol trinitrate (GTN 1 x 10-6 mol/L) was investigated in all groups.

Statistical analysis
Results are expressed as mean ± standard error of the mean (SEM) for n determinations. Changes in variables of each group were assessed by Friedmann two-way ANOVA followed by Wilcoxon matched-pairs signed-rank test. When corresponding variables of different groups were compared, the Mann-Whitney U-test was used. Differences were considered significant only if p < 0.05.

Drugs
Acetylcholine and EDTA were obtained from Sigma chemical (Deisenhofen, Germany), glycerol trinitrate from Schwarz Pharma (Monheim, Germany), dextran and Ficoll hypaque from Pharmacia Biotech (Freiburg, Germany), PBS from Life Technologies (Eggenstein, Germany), and sodium citrate from Merck (Darmstadt, Germany).

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Hemodynamic changes
Changes in left ventricular contractile parameters, heart ate, and coronary flow rates in groups I–IV are shown in Table 1. Baseline characteristics of these hemodynamic parameters were comparable in the 4 groups (differences not statistically different). At the end of the reperfusion period, left ventricular contractile parameters in groups I and III recovered completely. However, in groups II and IV, a moderate but significant (p< 0.05) decrease in LVP was ascertained at the end of the reperfusion compared to group I. In these groups, the recovery of LVdP/dt_max and LVdP/dt_min was slightly attenuated without reaching significance. The recovery of the coronary flow was not statistically different between the 3 groups.

View larger version (34K): [in this window] [in a new window]   Fig 2. Increase in coronary flow (%) in isolated hearts in group I (A), group II (B), group III (C), and group IV (D) before ischemia (30 minutes), and after reperfusion induced by acetylcholine and glycerol trinitrate. The respective vasodilator was intracoronarily infused (1% of coronary flow).

	Comment

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The hallmark of ischemia-reperfusion injury is early endothelial dysfunction that occurs within a few minutes of reperfusion despite absence of ultrastructural damage [4]. This endothelial dysfunction manifests in a decrease of NO release [8, 9]. The reduced NO level mediates subsequent upregulation of cell adhesion molecules, which can enhance the interaction of leukocytes with the coronary endothelium [9, 16]. On the other hand, NO is inactivated by superoxide radicals released by activated leukocytes [17]. Activated PMNs induce a vasocontraction in rabbit aorta [7], and cat coronary arteries [6] mediated by inactivation of basal EDRF-release by free radicals. Neutrophil activation is a prerequisite for cell adhesion and margination in the vessel wall and contributes to reperfusion injury by a further release of oxygen free radicals and cytotoxic enzymes. Numerous mediators, including arachidonic acid metabolites, cytokines, anaphylatoxins, and oxidants, contribute to reperfusion-induced neutrophil activation and adhesion [18, 19]. In addition, there is compelling evidence that myocardial ischemia is associated with an activation of the complement system and that this process promotes further cardiac damage by inducing a series of inflammatory events, including chemotaxis and activation of PMNs [15, 20].

Although it is generally accepted that activated leukocytes contribute to endothelial dysfunction after myocardial ischemia by inactivating basal NO release, the role of plasma factors, most likely complement, in endothelial dysfunction was not yet clear. Using an isolated rabbit heart preparation we were able to define the effects of autologous PMNs or autologous plasma administered after global ischemia during reperfusion on postischemic recovery of coronary flow and endothelial dependent vasodilation. To determine whether the observed impairment of vasorelaxation was endothelial dependent or represented a generalized inhibition of vasodilation, we also tested the ability of the isolated heart to dilate to glycerol trinitrate. Figure 2 summarizes these results and compares them with the acetylcholine responses.

The underlying mechanism of this plasma dependent leukocyte-mediated endothelial dysfunction during reperfusion remains to be elucidated. It may be hypothesized that plasma induces an activation of PMNs that then release free radicals and cytotoxic enzymes. On the other hand, plasma factors may activate the coronary endothelium with subsequent translocation of adhesion molecules that initiate adhesion, migration, and activation of the polymorphonuclear leukocytes [8, 21, 22].

The nature of the involved plasma factors may be discussed. There is substantial evidence that an activation of the complement cascade occurs during myocardial ischemia and may be an essential step in the recruitment and activation of polymorphonuclear neutrophils [10]. C5a expresses potent chemotactic activity for neutrophils and releases granular enzymes as well as superoxide anions (O₂^-) and it has been shown that complement-activated granulocytes reduce coronary flow in the nonischemic pig heart [23]. In addition to the complement system, cytokines may be involved in mediating the observed endothelial dysfunction in our model. A role of TNF- in conjunction with PAF in ischemia/reperfusion injury has been proposed [19]. In addition, TNF- is a mediator of endothelial dysfunction observed after reperfusion of an ischemic vascular bed [24].

Interestingly, in contrast to the observed endothelial dysfunction, our study failed to show a contribution of PMNs alone toward postischemic myocardial contractile dysfunction. The recovery of left ventricular pressure after ischemia and reperfusion was attenuated only when plasma was administered either alone or in combination with PMNs during the reperfusion period. Depending on the experimental setup used, both a contribution of PMNs and plasma toward postischemic myocardial contractile dysfunction [15] and a lack of PMN contribution to reperfusion injury have been reported [25]. Species differences and also distinct experimental set-ups may explain these discrepancies.

In summary, the results of the present study suggest that reperfusion-induced endothelial dysfunction may be dependent on an interplay between plasma and PMNs. Neither administration of PMNs nor plasma alone induces an impairment in endothelial function during reperfusion and accordingly plasma factors may play a role in direct activation of neutrophils. Identification of the involved plasma factors could allow the development of more effective therapeutic strategies against postischemic endothelial dysfunction.

	Acknowledgments

 
We gratefully acknowledge the technical assistance of Heidi Hess, Christiane Gögelein, Adelheit Gatzke, and Angelika Westphal who performed many of these experiments.

	References

Top Abstract Introduction Material and methods Results Comment References

 

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