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Cardiovascular Research 1997 36(3):386-395; doi:10.1016/S0008-6363(97)00191-0
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

ACE-inhibition prevents postischemic coronary leukocyte adhesion and leukocyte-dependent reperfusion injury

Christian Kupatta, Helmut Habazettlb, Stefan Zahlera, Christian Weberc, Bernhard F Beckera,*, Konrad Messmerb and Eckehart Gerlacha

aDepartment of Physiology, Ludwig-Maximilians-University, Pettenkoferstr. 12, 80336 Munich, Germany
bInstitute for Surgical Research, University of Munich, Munich, Germany
cInstitute for Cardiovascular Disease, University of Munich, Munich, Germany

* Corresponding author. Tel. (+49-89) 5996402; Fax (+49-89) 5996378; E-mail u721101@sunmail.lrz-muenchen.de

Received 17 March 1997; accepted 18 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: Polymorphonuclear leukocytes (PMN), retained in the microvascular bed, can contribute to postischemic myocardial reperfusion injury. Since a beneficial effect of ACE-inhibition on reperfusion injury has been reported, we investigated the impact of cilazaprilat on PMN dependent reperfusion injury in isolated guinea pig hearts. Methods: Hearts (n = 5 per group) were subjected to 15 min of ischemia. Immediately thereafter, a bolus of PMN was injected into the coronary system. External heart work (EHW) and total cardiac nitric oxide release were measured. For microscopic evaluation, hearts received rhodamine 6G labelled PMN after ischemia, were arrested 5 min later and further perfused with FITC dextran (0.1%). Localization of retained PMN was assessed by fluorescence microscopy. Leukocyte activation was studied by FACS analysis of the adhesion molecule CD11b before and after coronary passage of the PMN. The ACE-inhibitor cilazaprilat (Cila, 2 µM) and the NO-synthase inhibitor nitro-L-arginine (NOLAG, 10 µM) were used to modulate nitric oxide formation of the heart. Results: Postischemic EHW recovered to 67±5% (controls) and 64±6% (Cila) of the preischemic value. Addition of PMN severely depressed recovery of EHW (39±2%) and NO release (39±6% of the preischemic value). Simultaneously, ischemia led to a substantial increase in postcapillary PMN adhesion (from 21±5 to 172±27 PMN/mm2 surface) and CD11b-expression of the recovered PMN (3-fold). Cila attenuated postischemic PMN adhesion (83±52 PMN/mm2) and activation of PMN, whereas it improved recovery of work performance (64±4%) and NO release (65±4%) in the presence of PMN. Conversely, NOLAG increased PMN adhesion (284±40 PMN/mm2) and myocardial injury. We conclude that ACE-inhibition prevents leukocyte dependent reperfusion injury mainly by inhibition of postcapillary leukocyte adhesion. The effect may be mediated by NO, given the proadhesive effect of NOLAG.

KEYWORDS Cilazaprilat; Coronary microcirculation; Fluorescence microscopy; Leukocyte adhesion; Myocardial stunning; Nitric oxide


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Reperfusion of an occluded coronary artery is the only known therapeutic strategy preventing myocardial necrosis if applied in time. Nevertheless, reperfusion itself can induce a certain damage, i.e. reperfusion injury. Clinically it may appear as reversible decrease of myocardial contractility (stunning) [1, 2], or as reversible loss of endothelial function [3]. However, both deficits may persist in single cases, if microcirculatory perfusion is impaired for a prolonged time, as indicated by low-reflow or no-reflow phenomena [4, 5].

Polymorphonuclear granulocytes (PMN) which are recruited and activated in the reperfused myocardium can contribute to myocardial reperfusion injury in patients [6], as well as in a variety of models in vivo [7–11]and in situ [12, 13]. Two alternative mechanisms are considered relevant for PMN retention in microcirculatory beds. Capillary plugging of PMN as physical process is caused by narrowing of capillary lumina and/or reduced leukocyte elasticity [14]. On the other hand, neutrophils may selectively interact with endothelial cells through adhesion molecules, as demonstrated after myocardial ischemia [15, 16]. The adhesion process of PMN on endothelial cells generally follows a two-step pattern [17], a slow movement of a leukocyte on the endothelial cell wall (rolling) and subsequent firm adhesion (sticking) (for review see [18])

Inhibition of the angiotensin I converting enzyme (ACE), which represents a widely used pharmacologic intervention in patients with coronary artery disease, was beneficial against myocardial reperfusion injury in several distinct models [19–23]. ACE-inhibition has been shown to reduce bradykinin degradation, in turn stimulating nitric oxide synthase [24, 25]. Since NO mitigates PMN adhesion in other vascular beds [26], we wanted to investigate the cardioprotective potential of ACE-inhibition during postischemic reperfusion in an isolated heart model where the contribution of PMN to reperfusion injury can be distinguished from other sources of postischemic damage. To evaluate the effect, we assessed pre- and postischemic myocardial and endothelial function in terms of external heart work and endothelial nitric oxide release, respectively. In addition, we performed in situ fluorescence microscopy to characterize the precise localization and quantity of postischemically retained PMN. To determine the importance of NO, ACE inhibition was consistently compared with a nitric oxide synthase inhibitor (NOLAG) in further experiments.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Materials
Cilazaprilat, which was a gift of Hofmann-La Roche (Basel, Switzerland), was used at 2 µM. Nitro-L-arginine (NOLAG, Sigma Chemie, Deisenhofen, FRG) was used at 10 µM, rhodamine 6G (Sigma Chemie) was used to label PMN. Percoll for PMN separation and fluorescein isothiocyanate–dextran (FITC dextran, MW 150 000) were purchased from Pharmacia Biotech AB, Uppsala, Sweden. Monoclonal antibodies against CD15 (Miltenyi, Cologne, FRG) were used for human PMN separation, whereas CD11b analysis was performed with monoclonal antibodies from Exalpha (Boston, MA).

2.2 PMN preparation
2.2.1 Homologous
For in situ microscopy experiments, PMN (guinea pig) were isolated as previously described [27, 28]. Using HES sedimentation, Percoll separation and red cell lysis with distilled water, a pellet of at least 95% pure, 99% viable PMN was obtained. PMN were incubated with rhodamine 6G (0.005%, 10 min), counted (Coulter Counter ZM with Channelyzer, Coulter Electronics, King, NC, USA), and adjusted to the required cell content per ml (10x106/ml).

2.2.2 Human
In order to study PMN activation by flow-cytometrical surface analysis, a heterologous source of PMN was used, because no guinea pig antibody against CD11b was available to the best of our knowledge. Human PMN were isolated from peripheral venous blood of healthy volunteers by magnetic bead separation, as described elsewhere [29]. Briefly, purification of neutrophils from 10 ml human blood (EDTA 0.1%) was achieved by magnetic separation of cells previously labelled with a CD15 antibody carrying iron microbeads (Miltenyi Biotech, Bergisch Gladbach, FRG). Cells were analyzed by flow cytometry (FACScan, Becton Dikinson, San Jose, CA, USA) and proved to be 99% PMN. Cells were resuspended in Tyrode's solution (pH 7.40), counted and diluted to 10x106 cells/ml. As established in an earlier study, the cells prepared by these two techniques are not prestimulated and of comparable reactivity [29].

2.3 Heart preparation
The care of the animals and all experimental procedures conform with the Guide for the Care and Use of Laboratory Animals. Guinea pig hearts were isolated and perfused as previously described. Briefly, animals (male, 200–250 g) were anesthetized (Ketanest 200 mg/kg body weight) and decapitated. The hearts were arrested with cold saline and removed quickly from the thorax. The aorta was canulated and retrogradely perfused with a modified Krebs–Henseleit buffer gassed with 97% O2 and 3% CO2 (37°C, pH 7.40).

For microscopic evaluation (Langendorff preparation) caval, azygos and pulmonary veins were ligated. Retrograde perfusion was maintained throughout the experiment and in situ microscopic period.

The working heart preparation was performed, as previously described [30]. The caval and azygos veins were ligated. Pulmonary venous entry was used for a canula which, in the working mode of the heart, provided orthograde access for the perfusate. In the perfusion apparatus it was possible to switch between non-working (Langendorff) mode and working heart mode. External heart work was calculated as the sum of pressure–volume work (developed aortic pressurexcardiac output) and acceleration work [29].

2.4 Experimental protocols
Immediately after canulation, hearts were perfused at constant aortic pressure (80 cm H2O) for 5 min to finish preparation. At this point, hearts were assigned either to microscopic evaluation or to functional studies including determination of external heart work, nitric oxide release and leukocyte activation.

In experiments assessing external heart work, pressure volume work was performed (preload 12 cm H2O, afterload 60 mmHg) for 20 min (W1, see Fig. 1A), followed by 10 min of constant-flow Langendorff perfusion (5 ml/min). After ischemia (15 min, 37°C), hearts were initially reperfused in the non-working Langendorff mode (5 ml/min, 20 min) before a second period of external heart work at the given conditions was begun.


Figure 1
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  		Fig. 1 L1=Langendorff period 1 (5 ml/min volume constant perfusion); L2=Langendorff period 2 (5 ml/min); W1=Working period 1; W2=Working period 2; PMN=post-ischemic application of 107 PMN.

 
For microscopic evaluation, perfusion was continued at constant coronary flow of 5 ml/min for 30 min (see Fig. 1B). Usually, global ischemia was next imposed for 15 min, during which hearts were immersed in warm perfusate (37°C, pH 7.40). Thereafter, reperfusion was started, again at 5 ml/min, lasting for 5 min in all experiments. PMN were applied when reperfusion was started and recovered in the coronary effluent for 3 min for further analysis. Cold cardioplegic solution was then infused to arrest the heart. After being placed on a stage displaying the left ventricle area to the microscope, FITC-infusion was begun.

Most experimental series involved 10 hearts, 5 of each used in parallel experiments for either microscopic or functional evaluation. Time matched ‘control’ experiments were performed without ischemia, 107 PMN being applied after 45 min of perfusion. In the ‘ischemia’ series PMN were infused in the first min of reperfusion after 15 min of ischemia. In addition, one working heart group was subjected to ischemia without PMN infusion. To assess the role of endogenous NO, cilazaprilat (2 µmol/l) and nitro-L-arginine (NOLAG, 10 µmol/l) were added to the perfusate in order to stimulate (indirectly) or inhibit NO synthase activity in experiments including ischemia.

2.4.1 Nitric oxide release
In working heart experiments, a nitric oxide sensitive, microtip probe (WPI, Sarasota, FL, USA) was placed into the pulmonary artery directly behind the pulmonary valve, the tip of the NO sensor being inserted into a T-tube. Isothermal conditions were achieved by immersing preparations in Tyrode's solution at 37°C. Validation of the probe recording in the coronary effluent was performed using bradykinin to generate NO by the endothelium, and erythrocyte-borne hemoglobin (hematocrit 5%) to sequester NO (data not shown). Reproducible stimulation and suppression of the signal to zero were observed, respectively.

Considerable variability of basal release of NO between hearts during preischemic volume constant perfusion (L1) was found in all groups. Comparability of volume constant perfusion and working heart mode (different flow rates) was achieved by calculating NO release per minute per g dry weight of the hearts from the concentration data. Postischemic NO release is given in percent of preischemic values.

2.4.2 Fluorescence microscopy
For the purpose of in situ microscopy hearts were arrested by cold cardioplegic solution (potassium 28 mmol/l). Thereafter they were placed on a specially designed microscopic stage holder, the surface of the left ventricle being exposed to a microscope (Ploemopak, Leitz, Wetzlar, FRG) with a 10-fold objective (L10, 0.22 Aperture, Leitz). Images were generated by a charge coupled device camera (COHU 4400, Prospective Measurements, San Diego, CA). To analyze vessel distribution fluorescein isothiocyanate dextran (FITC 0.1%) was infused (15 ml/h) and excited by epi-illumination with a H130 mercury light source and filtered with an I2,3 filter block (Leitz, excitation/emission spectra: 450–490/>515 nm). The distribution of PMN was studied under a rhodamine 6G selective N2 filter block (Leitz, excitation/emission spectra: 515–560/>580 nm).

This method allowed for localization of PMN with respect to the vascular lumen. Capillary plugging was assessed by counting PMN in regions devoid of venules. 5 fields with an area of 1.2 mm2 each were studied per heart. Postcapillary venular adhesion was observed in venules of 10–50 µm diameter (small venules, n = 8–10 per heart). In addition, venules of 50–300 µm diameter (large venules, n = 4–5 per heart) were investigated. Fig. 2 demonstrates FITC and rhodamine fluorescence of an epicardial surface of a postischemic heart, showing substantial leukocyte adhesion in venules, but not in arterioles.


Figure 2
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  		Fig. 2 Example of PMN retention in reperfused epicardial vessels. Scale bar: 100 µm. The upper panel demonstrates distribution of vessels (FITC–dextran 0.1%), two branches of arteriolae marked by white arrows. Lower panel shows pattern of leukocyte retention. Plenty of PMN are localized in venular vessel lumina, while no adhesion was found in arteriolae.

 
2.4.3 Flow cytometry analysis
To further assess the degree of activation induced during coronary passage, PMN were analyzed in a flow cytometer (FACScan, Becton Dickinson, San Jose, CA, USA) using a phycoerythrin (PE) fluorescence-labelled monoclonal CD11b-antibody (Moab) which bound to the PMN epitope CD11b, part of the heterodimeric adhesion molecule CD11b/CD18 (MAC-1). Forwardscatter analysis and gating of the PMN range provided analysis of a homogeneous PMN population. Isotype controls with a nonbinding, fluorescence labelled Moab (anti-mouse IgG1) served to determine nonspecific binding and background fluorescence. Activation of PMN was expressed in relation to the basal values before coronary passage. Data analysis of 10 000 events was performed with Lysis II software on a Hewlett Pakard system.

2.5 Statistical methods
The results are given as mean±SEM. Statistical analysis was performed with one way analysis of variance (ANOVA). Whenever a significant effect was obtained with ANOVA, we performed multiple comparison tests between the groups using Dunn's test (Sigmastat statistical program). Differences between groups were considered significant for p<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
Since beneficial effects of ACE-inhibitors have been demonstrated following myocardial hypoperfusion in vivo, we studied the effects of cilazaprilat on PMN dependent myocardial reperfusion injury.

3.1 Functional reperfusion injury
3.1.1 Coronary and myocardial function
At a concentration of 2 µM, cilazaprilat evoked no change in coronary flow (CF) or coronary perfusion pressure (CPP) preischemically (Table 1), in contrast to NOLAG which, at 10 µM, elevated CPP. After ischemia, CPP first decreased during postischemic dilation, before rising above preischemic values in all groups. Fittingly, CF in W2 was significantly suppressed vs. the preischemic value in all groups (Table 1). The same tendency existed in the presence of cilazaprilat, excluding a major hemodynamic effect of the drug. In contrast, NOLAG blunted the postischemic dilation and decreased CF in W2 more extensively than PMN alone.


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  		Table 1 Hemodynamic characterization of the experimental groups

 
Values of external heart work prior to ischemia for the various experimental groups did not differ significantly (Table 1). Application of a PMN bolus without ischemia caused only a slight detriment of external heart work (Control PMN, Fig. 3). After ischemia alone, recovery of EHW deteriorated to 67±5% of the initial value. Ischemia and a PMN bolus acted synergistically, reducing recovery of EHW to 39±6%. This level was further decreased to 29±6% in the presence of NOLAG (1 µM). If the concentration of NOLAG was increased to 10 µM, no recovery could be measured due to ventricular fibrillation and pump failure. Cilazaprilat (2 µM), however, counteracted the detrimental effects of PMN without (64±4%) or in the presence of NOLAG (1 µM, 41±3%), whereas it did not influence the recovery of reperfused hearts not confronted with PMN (65±4% recovery).


Figure 3
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  		Fig. 3 Recovery of external heart work in Working Phase 2, given in percent of results in Working Phase 1. Control PMN=time matched control experiments. Ischemia = 15 min of warm ischemia, Ischemia+PMN = bolus of 5x106 PMN given with the beginning of reperfusion, Ischemia+NOLAG+PMN = postischemic PMN bolus in the presence of 1 µM Nolag. Ischemia+NOLAG+Cila+PMN = postischemic PMN bolus in the presence of 1 µM Nolag and 2 µM cilazaprilat. Ischemia+Cila+PMN = postischemic PMN bolus in the presence of cilazaprilat. Ischemia+Cila = Ischemia and reperfusion in the presence of cilazaprilat.

 
Thus, postischemic myocardial performance was related to the absence or presence of PMN. Cilazaprilat effectively prevented PMN-induced loss of function, NOLAG augmented it.

3.1.2 Studies on nitric oxide release
Release of nitric oxide is a specific feature of the endothelium, regulating vascular resistance. We investigated endothelial integrity by measuring NO release of the whole coronary system. Isolated guinea pig hearts performing pressure–volume work prior to ischemic challenge (W1) released NO into the coronary effluent at variable, but not significantly different rates (Table 1). The concentration approximated 2–3 µM. Preischemic ‘basal’ nitric oxide release in hearts perfused in the Langendorff mode (L1) was markedly lower than during work (W1, Table 2). Surprisingly, cilazaprilat had no discernible effect on preischemic NO release, as already suggested by coronary hemodynamics (Table 1). Since no in situ calibration of the NO-flow probe was possible in the presence of NOLAG, no quantitative data were obtained for these experimental groups. In hearts receiving PMN without preceding ischemia (control+PMN), the capability to form NO was only slightly attenuated over time, the difference between L1 and L2 amounting to 0.5 nmol/min/g (results not shown).


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  		Table 2 Nitric oxide release prior to ischemia in the different experimental groups

 
To correct for differences of NO release due to the mode of perfusion, postischemic values of L2 or W2 in Fig. 4 are given in percent of the preischemic values obtained in the same mode (L1 or W1). In hearts subjected to ischemia, a transient increase in NO release after 5 min of reperfusion was displayed in most groups, which was most prominent in the presence of cilazaprilat (Fig. 4). Although the postischemic increase in NO release lasted only a few minutes, it was during this period that PMN application into the coronaries occurred in the present experiments. Over the subsequent 20 min, NO release decreased by 30% on average. The drop was found to be most pronounced in the PMN group (46±1% of the preischemic value). This leukocyte-dependent effect was prevented by cilazaprilat, whereas no effect of cilazaprilat was detected in hearts not subjected to PMN infusion. Experiments with inhibition of NO synthases (NOLAG, 10 µM) displayed neither an increase after 5 min nor a decrease after 20 min of reperfusion (data not shown). However, the lack of reaction of the probe after challenge with an erythrocyte bolus suggested that no endogenous NO formation was taking place under this condition anyway.


Figure 4
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  		Fig. 4 Coronary nitric oxide release measured by an NO-flow probe (WPI) in strict isothermia (37°C). Values are given in percent of preischemic level (see Table 2). Ischemia = 15 min of warm ischemia, PMN = bolus of 5x106 PMN given with the beginning of reperfusion, Cila = 2 µM Cilazaprilat. §=p<0.05 v. 5 min after ischemia; #=p<0.05 v. cilazaprilat groups at the same time.

 
In work phase 2, hearts released only about half (55±0.5%) of the NO detected in W1 (Fig. 4). Hearts in the ischemia PMN group released less nitric oxide than in the absence of PMN. Pretreatment with cilazaprilat prevented this effect of PMN but did not restore postischemic NO release to the preischemic level. The relative conservation of the postischemic NO release through cilazaprilat was not related to a higher level of NO preischemically (Table 1).

3.2 Leukocyte adhesion and activation after ischemia
3.2.1 Capillary plugging
PMN can be trapped in the microcirculatory system through plugging in capillaries, where the vessel diameter is reduced to about 10 µm and less. As shown in one example (Fig. 5), ischemia induced some plugging of PMN in epicardial capillaries. This impression was confirmed in quantitative analysis of 5 capillary fields per heart (5 hearts per group, Fig. 6). While cilazaprilat did not affect postischemic plugging, NOLAG further increased it.


Figure 5
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  		Fig. 5 Example of postischemic PMN plugging in capillaries (Ischemia group). Upper Panel: FITC–fluorescence picture, 1.2 mm2 field. Lower Panel: Rhodamine 6G fluorescence picture showing PMN in capillaries.

 

Figure 6
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  		Fig. 6 PMN plugging in capillaries of reperfused hearts. Control = no ischemia; Ischemia = 15 min of warm ischemia; Cila = 2 µM Cilazaprilat, NOLAG = 10 µM nitro-L-arginine. Mean±SEM. *=p<0.05 v. all other groups.

 
3.2.2 Postcapillary adhesion
3.2.2.1 Small venules
Postcapillary venular adhesion of PMN was found to be 21±5 PMN per mm2 vessel surface area in control experiments. Ischemia induced a marked increase in postcapillary adhesion (Fig. 7). Cilazaprilat reversed this increase to a level not significantly different from control values. NOLAG, however, further increased the ischemia-dependent rise of postischemic adhesion to more than 13-fold the control level. Fig. 8A and B show two representative examples of postcapillary adhesion in small venules with either cilazaprilat (Fig. 8A) or NOLAG (Fig. 8B) added to the perfusate.


Figure 7
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  		Fig. 7 PMN adhesion in small and large venules in reperfused hearts. Control = no ischemia; Ischemia = 15 min of warm ischemia; Ischemia+Cila = 15 min of warm ischemia in the presence of 2 µM cilazaprilat, Ischemia+NOLAG = 15 min of ischemia in the presence of 10 µM nitro-L-arginine. Mean±SEM. *=p<0.05 v. paired small venule quantity, #=p<0.05 v. Control in small venules; +=p<0.05 v. Ischemia+Cila in small venules; §=p<0.05 v. Control in big venules.

 

Figure 8
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  		Fig. 8 Examples of postischemic PMN adhesion in small venules. Upper Panel: FITC–fluorescence picture, 1.2 mm2 field. Lower Panel: Rhodamine 6G fluorescence picture showing PMN in venules. A: Representative example of the Ischemia+Cila group; B: Representative example of the Ischemia+NOLAG group.

 
3.2.2.2 Large venules
Very few PMN were localized in large venules of control hearts (6±1 PMN per mm2 vessel surface area). Ischemia induced a rise of PMN adhesion in this class of venules to 37±8 PMN per mm2 surface area. Cilazaprilat slightly reduced this value (21±5 PMN per mm2 surface area), although no significant effect could be obtained. NOLAG, in contrast, tended to increase postischemic PMN adhesion in the large venules to 48±9 PMN per mm2 surface area (Fig. 7). Taken together, these findings suggest that postcapillary adhesion rather than capillary plugging accounts for increased coronary PMN retention after ischemia in the present model. ACE-inhibition reduces, while NOLAG increases adhesion, indicating an antiadhesive effect of nitric oxide.

3.2.3 Leukocyte activation
Activation of human leukocytes was further analyzed by quantifying CD11b surface expression. As shown in Fig. 9, PMN of control experiments (without ischemia) displayed almost no difference in CD11b surface expression before and after coronary passage. Postischemically, however, a 2.6 fold elevation of CD11b expression had occurred on PMN emerging from the coronary system. When cilazaprilat was present, this increase in CD11b-associated fluorescence intensity was completely abolished, whereas NOLAG further enhanced CD11b expression. These findings indicate that cilazaprilat prevents activation and subsequent postcapillary adhesion, most likely through a nitric oxide mediated mechanism.


Figure 9
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  		Fig. 9 Leukocyte activation by CD11b expression (FACScan, Becton Dickenson). PMN before coronary passage (hatched bars) and after coronary passage (solid bars) were analyzed. #=p<0.05 v. paired pre-passage value.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
Early implementation of ACE inhibition after myocardial infarction has proven beneficial in several models [19–23]. In search of a possible ‘local’ action to explain this effect, we have previously found ACE-inhibitors to mitigate postischemic functional damage in saline-perfused hearts via a bradykinin and, ultimately, nitric oxide mediated pathway [31]. NO, in turn, is known to mitigate adhesion of PMN in various vascular beds. Since PMN strongly exacerbate post-ischemic damage in the isolated heart model [27, 28, 31], we set out to investigate the simultaneous occurrence and interdependence of four phenomena: First, the ability of an ACE inhibitor (cilazaprilat) to alleviate postischemic loss of myocardial performance induced by PMN, as opposed to the action of an inhibitor of NO-synthase (nitro-L-arginine). Second, the actual effect of these interventions on postischemic coronary release of nitric oxide. Third, the corresponding changes in PMN adhesion according to specific localization in capillaries, small venules and collecting veins. Fourth, PMN activation during coronary passage in post-ischemic hearts in the presence of cilazaprilat or nitro-L-arginine.

4.1 Influence of ischemia and reperfusion
As a result of ischemia and reperfusion alone, isolated hearts suffered from compromised external heart work (Fig. 3), i.e. myocardial stunning, and a general decline in production of NO (Fig. 4). Both phenomena were aggravated by application of PMN during reperfusion. Reactive oxygen species play a crucial role in this type of reperfusion injury, as shown by others [32]as well as in our model [27, 28]. Beyond an inherent detrimental effect on cellular processes, reactive oxygen species may enhance interactions between endothelium and PMN by externalization of stored P-selectin and PAF [29, 33]as well as by activation and translocalization of the leukocyte receptor Mac-1 (CD11b/CD18) [34]. The latter parameter was the one we chose to observe (Fig. 9). Indeed, we found CD11b expression to be markedly elevated on PMN after passage through the coronary system of post-ischemic hearts. These effects may explain the fast and significant rise of venular leukocyte adhesion immediately after resumption of coronary perfusion, a time at which de novo synthesis of adhesion molecules cannot yet have taken place. We cannot rule out a contribution of plugging leukocytes, which has approximately doubled postischemically (Fig. 6), although the myocardial stunning and loss of endothelial NO-release after ischemia (Figs. 3 and 4Go) fits best to the steep increase of postcapillary PMN adhesion (Fig. 7).

4.2 Influence of ACE-inhibition on PMN dependent reperfusion injury
Several studies have shown a cardioprotective effect of ACE-inhibition after limited ischemia in vivo and in situ [19–23]. However, this work for the first time presents direct microscopic observations that ACE-inhibition by cilazaprilat can acutely reduce ischemia-induced PMN adhesion and activation in the coronary venules independent of any coronary hemodynamic influence. In parallel experiments, cilazaprilat was capable of blunting PMN dependent loss of myocardial (external heart work) and endothelial function (reduced production of NO).

In the presence of the ACE-inhibitor ramiprilat, Schoelkens and coworkers found an increase in bradykinin concentration immediately after ischemia in isolated rat hearts [35]. Decreased bradykinin degradation due to ACE inhibition leads to a stimulation of endothelial nitric oxide synthase [31]. In this context it is important to point out that NO may act as an antioxidant and radical scavenger, which may interfere with the reperfusion-induced release of oxygen free radicals and, thus, prevent subsequent activation of endothelial cells and PMN [36]. The dependence on NO is made even more probable by the fact that cilazaprilat does not exert a radical scavenging effect itself (data not shown), in contrast to e.g. captopril [37]. With the concentration of cilazaprilat used (2 µM), no effects were seen on recovery of external heart work during cell-free reperfusion, as opposed to findings with higher concentrations of a similar drug [24]. The negative effect of NOLAG on myocardial recovery is counteracted by cilazaprilat (Fig. 3) suggesting that in fact cilazaprilat really does activate nitric oxide synthase. Interestingly, NO release was maintained by cilazaprilat at a higher level even after PMN application, i.e., the time at which adherent PMN produce tissue damaging radicals and oxidants.

How does the observed impact of cilazaprilat on myocardial and endothelial recovery relate to PMN retention of the hearts? A multitude of factors is able to change the balance of pro- and antiadhesive behaviour of coronary endothelial cells, notably NO [26]. In the presence of cilazaprilat, we observed slightly increased nitric oxide release during early reperfusion, the period in which PMN were injected (Fig. 4). Indeed, PMN, given in the early reperfusion, adhered less in the presence of cilazaprilat (Fig. 7), while, in contrast, capillary plugging was barely influenced (Fig. 6). At this point, however, it cannot be ruled out that nitric oxide formation in the presence of cilazaprilat has additional, indirect effects inhibiting proadhesive mediators formed during reperfusion, e.g. endothelin-1 [38, 39].

Since NO release from the whole intact heart is a ‘net’ effect of nitric oxide production and consumption, the local increase of NO might have been higher but, in part, consumed by oxygen free radicals. The crucial importance of NO for leukocyte adhesion in our model is demonstrated by application of NOLAG, which increased adhesion and activation of PMN (Figs. 8 and 9Go). NOLAG is a nonspecific NO synthase inhibitor which affects endothelial (eNOS) and inducible nitric oxide synthase (iNOS). However, in primary cultured endothelial cells from isolated rat hearts, we could not detect iNOS by Western blotting (data not shown). Therefore NOLAG most likely affects the eNOS, the same enzyme which is activated in the presence of cilazaprilat.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
In summary, we have investigated the influence of ACE-inhibition on PMN adhesion and myocardial and endothelial function after a PMN bolus given early in reperfusion of isolated hearts. Cilazaprilat mitigated postischemic increase in PMN adhesion, foremost in postcapillary venules. Concomitantly, PMN-induced loss of external heart work and reduction of endothelial nitric oxide release were prevented by cilazaprilat. ACE-inhibition also prevented activation of PMN during passage of the coronary system of postischemic hearts. Inhibition of NO-synthase caused the exact opposite in each case. Thus, the protective effects of ACE inhibition may well be mediated by the oxygen radical scavenging ability of NO and evolve from reduced adhesion of PMN in the presence of NO.

The findings of the present study underscore the importance of leukocyte adhesion and activation after ischemia. Pharmacological interventions aimed at reducing leukocyte induced reperfusion injury may well benefit those patients who already suffer from compromised myocardial contractility. Recent clinical investigations showing leukocyte activation in man as a potential risk factor of recurrent coronary events enforce this approach [40].

Time for primary review 51 days.


   Acknowledgements
 
Cilazaprilat was a gift of Hofmann La Roche (Basel, Switzerland), which also provided the WPI nitric oxide sensor. The study was supported in part by the Friedrich-Baur-Foundation of the University of Munich. We thank Veronika Frei for expert technical assistance.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

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