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Cardiovascular Research 2000 47(2):294-305; doi:10.1016/S0008-6363(00)00115-2
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Comparative study of AMP579 and adenosine in inhibition of neutrophil-mediated vascular and myocardial injury during 24 h of reperfusion

Jason M Buddea, Daniel A Veleza, Zhi-Qing Zhaoa,*, Kenneth L Clarkb, Cullen D Morrisa, Satoshi Murakia, Robert A Guytona and Jakob Vinten-Johansena

aCardiothoracic Research Laboratory, The Carlyle Fraser Heart Center/Crawford Long Hospital, Emory University School of Medicine, 550 Peachtree St., NE, Atlanta, GA 30365-2225, USA
bRhône-Poulenc Rorer Research and Development, Collegeville, PA 19426, USA

* Corresponding author. Tel.: +1-404-686-2511; fax: +1-404-686-4888 zzhao{at}emory.edu

Received 21 February 2000; accepted 25 April 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Specific methodology
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Objective: The purpose of this study was to compare protective effects of AMP579 and adenosine (Ado) at reperfusion (R) on inhibition of polymorphonuclear neutrophil (PMN) activation, PMN-mediated injury to coronary artery endothelium, and final infarct size. Methods: In anesthetized dogs, 1 h of left anterior descending coronary artery occlusion was followed by 24 h R and drugs were administered at R. Control (n=8, saline control), AMPI (n=7, AMP579, 50 µg/kg i.v. bolus followed by 3 µg/kg/min for 2 h), AMPII (n=7, AMP579, 50 µg/kg i.v. bolus), AMPIII (n=7, AMP579, 3 µg/kg/min i.v. for 2 h), and Ado (n=7, adenosine, 140 µg/kg/min i.v. for 2 h). Results: AMP579 in vitro directly inhibited superoxide radical (O–2) generation (nM/5x106 PMNs) from PMNs dose-dependently (from 17±1* at 10 nM to 2±0.2* at 10 µM vs. activated 30±2). However, inhibition of O–2 generation by Ado at each concentration was significantly less than for AMP579. The IC50 value for AMP579 (0.09±0.02 µM) on O–2 generation was significantly less than that of Ado (3.9±1.1 µM). Adherence of unstimulated PMN to postischemic coronary artery endothelium (PMNs/mm2) was attenuated in AMPI and AMPIII vs. Control (60±3* and 58±3* vs. Control 110±4), while Ado partially attenuated PMN adherence (98±3*). Accordingly, endothelial-dependent vascular relaxation was significantly greater in AMPI and AMPIII vs. Ado. At 24 h R, myocardial blood flow (MBF, ml/min/g) in the area at risk (AAR), confirmed by colored microspheres, in AMPI and AMPIII was significantly improved (0.8±0.1* and 0.7±0.1* vs. Control 0.3±0.04). Infarct size (IS, TTC staining) in AMPI and AMPIII was significantly reduced from 38±3% in Control to 21±4%* and 22±3%*, respectively, confirmed by lower plasma creatine kinase activity (I.U./g protein) in these two groups (27±6* and 32±2* vs. 49±3). Cardiac myeloperoxidase activity (MPO, Abs/min) in the AAR was significantly reduced in AMPI and AMPIII vs. Control (36±11* and 35±10* vs. 89±10). However, changes in MBF, IS and MPO were not significantly altered by Ado. Conclusions: These data suggest that continuous infusion of AMP579 at R is more potent than adenosine in attenuating R injury, and AMP579-induced cardioprotection involves inhibition of PMN-induced vascular and myocardial tissue injury. *P<0.05 vs. Control.

KEYWORDS Adenosine; Free radicals; Ischemia; Leucocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Specific methodology
 3 Results
 4 Discussion
 5 Conclusion
 References
 
Abundant evidence from both experimental animals and clinical observations indicate that reperfusion-induced myocardial injury is a problem encountered by all therapeutic approaches to the treatment of acute ischemic heart disease [1–4]. Studies have suggested that polymorphonuclear neutrophil (PMN) activation and PMN-mediated inflammatory responses play major roles in ischemia–reperfusion-induced myocardial injury [5–8]. Potential mechanisms underlying PMN-mediated myocardial injury during early reperfusion (<6 h) may include adherence to vascular endothelium to elicit PMN and endothelial cell interaction, and release a number of reactive oxygen species such as superoxide radicals, which directly induce activation and damage to endothelial cells. Mechanical plugging of PMNs in microvessels may also cause an increase in capillary blood flow resistance and thereby contribute to the ‘no-reflow’ phenomenon. As the reperfusion time increases, injury to myocytes may be mediated in part by direct cell–cell contact between migrated PMNs and myocytes [9–12]. To protect the heart from PMN-mediated myocardial injury, a number of studies have shown that administration of adenosine during reperfusion protects ischemia–reperfusion injury through modulation of PMN function [13–16]. Recently, we found that intra-atrial infusion of adenosine during reperfusion reduced myocardial injury by inhibiting PMN adherence to coronary artery endothelium, PMN accumulation in postischemic myocardium, and damage of endothelium-dependent vascular relaxation in a canine model of 1 h ischemia and 6 h of reperfusion [17]. However, a short half-life of adenosine (a few seconds) and a necessity for higher concentrations for effective inhibition of PMN activation [18,19] may limit its cardioprotective effect in in vivo models due to the progression of PMN-mediated injury during late reperfusion [12,20]. Therefore, an adenosine analog with a longer pharmacological half life, an effective action in the nanomolar range as well as a potent inhibitory effect on PMN activation compared to adenosine may help to efficiently eliminate ischemia–reperfusion-induced injury to the heart.

AMP579 (1S-[1a,2b,3b,4a(S*)]-4-[7-[[1-[(3-chloro-2-thienyl)methylpropyl]propyl-amino]-3H-imidazo[4,5-b] pyridyl-3-yl]-N-ethyl-2,3-dihydroxycyclopentane carboxamide) is a new adenosine analog with high affinities for the adenosine A1 (Ki=5 nM) and A2A (Ki=56 nM) receptor subtypes. Its half-life lasts approximately 1 h [21]. Studies have shown that the administration of AMP579 either during ischemia or prior to reperfusion reduces myocardial infarction in acute ischemic–reperfused models (less than 6 h of reperfusion) of rat, pig and dog [22–25]. AMP579 in vitro directly inhibits superoxide free radical generation from platelet activating factor (PAF)-activated canine PMNs, attenuates the adherence of canine PMN to thrombin-activated coronary artery endothelium, and protects PMN-mediated damage to coronary artery endothelium [26]. These results suggest that AMP579 may attenuate myocardial injury by inhibiting PMN-mediated inflammatory reactions in vivo. The purpose of this study was to determine whether exogenous AMP579 and adenosine during reperfusion limit the progression of myocardial injury after prolonged reperfusion by inhibiting PMN activation and cell–cell interaction between PMN and coronary vascular endothelium, preserving myocardial blood flow, and reducing infarct size in a canine closed-chest ischemia–reperfusion model.


   2 Specific methodology
Top
 Abstract
 1 Introduction
 2 Specific methodology
3 Results
 4 Discussion
 5 Conclusion
 References
 
2.1 In vivo studies
2.1.1 Surgical preparation of animals
The animals used in this study were maintained in accordance with the guidance of the committee on the Animal Welfare Act and Emory University Veterinary policies and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Council (Department of Health and Human Services publication No. NIH 85-23, revised 1985).

Dogs of either sex were used in the study. All animals were initially anesthetized with intramuscular morphine sulfate (4 mg/kg). A bolus injection of pentothal (20 mg/kg) was given followed by continuous inhalation of isoflurane (0.5–2% in oxygen) after endotracheal intubation. A left lateral thoracotomy was performed, and the pericardium was widely opened. Micromanometer pressure transducers were inserted into the left internal mammalian artery and ventricle to monitor aortic and ventricular pressure. A pair of ultrasonic crystals was implanted in the anterior-midmyocardium to measure regional contractile function. A doppler flow probe was placed around the proximal left anterior descending coronary artery (LAD) for measurement of coronary artery flow. A catheter was inserted into the left atrium for injection of colored microspheres to measure regional myocardial blood flow. The LAD just distal to the first diagonal branch was reversibly occluded completely by pulling up on the snare to produce a zone of regional ischemia in the left ventricle. After 2 h of reperfusion, the thoracotomy incision was closed in layers, and a regimen of broad spectrum antibiotics and analgesia was initiated throughout the reperfusion period.

2.1.2 General experimental protocol
All dogs were randomly assigned to one of five groups. Control (n=8, saline control), AMPI (n=7, AMP579, 50 µg/kg i.v. bolus followed by 3 µg/kg/min for 2 h), AMPII (n=7, AMP579, 50 µg/kg i.v. bolus), AMPIII (n=7, AMP579, 3 µg/kg/min for 2 h), and Ado (n=7, adenosine, 140 µg/kg/min for 2 h). In all experimental groups, the left coronary artery was occluded for 1 h of ischemia and released to begin 24 h of reperfusion. The ECG and regional confirmation of pressure-segment length loops were used initially to confirm the presence of myocardial ischemia, but the severity of blood flow reduction was measured by colored microspheres. Intravenous administration of AMP579 and Ado was started 5 min before reperfusion. Hemodynamic and regional contractile function were measured at baseline, ischemia, 5 min after drug infusion and just before reperfusion, 2 h (measurement taken just before AMP579 and Ado had been discontinued) and 24 h of reperfusion. At the end of 24 h of reperfusion, a blood sample was withdrawn for PMN isolation. Segments of LAD and left circumflex (LCX) coronary arteries were isolated and used to evaluate agonist-stimulated vascular endothelial response and to quantify adherence of unstimulated PMNs to coronary artery endothelium. Nonischemic and ischemic myocardial tissue were used to determine myeloperoxidase activity, myocardial blood flow, water content, and infarct size (see below).

2.1.3 Plasma creatine kinase (CK) activity
Arterial blood samples were withdrawn at baseline, at the end of ischemia, 2 and 24 h of reperfusion to measure CK activity. Heparinized samples were centrifuged at 2500 g and 4°C for 10 min. The plasma was drawn off and analyzed spectrophotometrically for CK activity according to the method of Rosalki (Sigma Diagnostics). Plasma CK activity was expressed as I.U./mg protein.

2.1.4 Determination of area at risk and infarct size
After completing the isolation of coronary arteries, Unisperse blue dye was injected into the aortic root to stain the normally perfused region blue and outline the area at risk. After excision, the left ventricle was cut into transverse slices. The area at risk was separated from the non-ischemic zone and incubated for 10 min in a 37°C 1% solution of triphenyltetrazolium chloride to differentiate necrotic (pale) from non-necrotic area at risk tissue. The gravimetric method was used to quantify infarct size [17].

2.1.5 Measurement of myeloperoxidase activity (MPO) in cardiac tissue
Tissue samples (200 mg for each) from nonischemic and ischemic zones were homogenized. After centrifugation, the supernatants were decanted and mixed with O-dianisodine dihydrochloride and H2O2 in phosphate buffer. The change in absorbance was measured spectrophotometrically at 460 nm. MPO activity was expressed as absorbance units/min [17].

2.1.6 Determination of regional myocardial blood flow
Colored microspheres were used to quantify myocardial blood flow. Samples of non-ischemic, ischemic subepicardial and subendocardial myocardium were placed in tared vials marked according to their anatomical location and staining pattern. Tissues and reference blood samples were analyzed in a Spectra Max 250 microplate reader spectrophotometer (Molecular Devices) as reported previously [17]. Results are expressed as ml/min/g tissue.

2.1.7 Determination of myocardial water content
At the end of the experiment, heart tissues from non-ischemic, ischemic subepicardial and subendocardial myocardium were separated. Myocardial edema formation was quantified by the calculation of myocardial water content: % H2O=[1–(dry weight/wet weight)]x100.

2.2 In vitro studies
2.2.1 PMN isolation
After initial anesthesia on the final day of the experiment, arterial blood (40 ml) from different groups was sampled, respectively. PMNs were isolated by the Ficoll-Hypaque density gradient technique. Cell preparation contained >95% PMNs and cell viability was >90% (trypan blue exclusion). Isolated PMNs were pharmacologically stimulated [19].

2.2.2 PMN superoxide radical (O2–) production
O2– production by PMNs was determined by measuring the O2– dismutase-inhibitable reduction of ferricytochrome C to ferrocytochrome C using a Spectra Max 250 microplate reader spectrophotometer. PMNs (5x106/ml) were suspended in Hanks balanced salt solution. PAF was used as a physiological activator of PMNs [19]. This assay was used to compare concentration responses of AMP579 and Ado.

2.2.3 PMN adherence to coronary artery endothelium (basal endothelial function)
Alteration in cell–cell interaction between PMN and coronary artery endothelium after reperfusion by AMP579 and Ado was assessed using PMNs labeled with Zynaxis PKH2 vital fluorescent dye (Zynaxis Cell Science, Malvern, PA, USA). After in vivo experiment, coronary artery segments were carefully opened and placed in cell culture dishes. Labeled PMNs (4x105 cells/ml) were added to the dishes, allowed to incubate for 15 min, removed and placed on glass slides. The numbers of PMNs adhering to the endothelial surface in six separate microscopic fields were counted under epifluorescence microscopy (490 nm excitation, 504 nm emission) [19].

2.2.4 Postischemic vascular ring reactivity
After completion of the experiment, LAD and LCX coronary artery segments were carefully isolated and placed into Radnoti tissue baths containing Krebs–Henseleit solution at 37°C. After stabilization, the coronary rings were subsequently preconstricted with thromboxane A2–mimetic U46619 [GenBank] (5 nM) and dilated in concentration–response fashion with the endothelium-dependent vasodilator, acetylcholine, and the endothelium-independent vasodilator, sodium nitroprusside in incremental concentrations. Responses to vasodilators were analyzed using a videographics program developed in our laboratory [19].

2.2.5 Criteria for exclusion
Standard exclusion criteria were (1) transmural myocardial blood flow in the area at risk during ischemia exceeding 0.15 ml/min/g tissue; (2) unclear demarcation of the area at risk after coronary occlusion by Unisperse blue staining; (3) ventricular fibrillation that did not convert to normal rhythm in 2 min by electric shock during reperfusion and (4) failure to complete the entire protocol. Forty-eight dogs were initially entered into the study, of which 36 are represented in the final analysis of the results. Of the twelve dogs that were excluded from the data analysis according to exclusion criteria, three were from the Control group, three from AMPI, one from AMPII, two from AMPIII and three from Ado groups.

2.3 Statistical analysis
Concentration–response curves of vascular relaxation were calculated as a percentage of U46619 [GenBank] -induced increase in isometric force. One-way analysis of variance and then Duncan's post-hoc test were used to analyze differences between such parameters as superoxide radical production, PMN adherence, vascular responses, MPO, myocardial blood flow and infarct size data. Hemodynamic, regional contractile function and other time-dependent determinations were analyzed by repeated analysis of variance. A P value <0.05 was accepted as statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Specific methodology
 3 Results
4 Discussion
 5 Conclusion
 References
 
3.1 Generation of O2– from activated PMNs
Inhibition of O2– generation from PMNs by AMP579 and Ado is shown in Fig. 1. AMP579 inhibited O2– production in a concentration-dependent manner. AMP579 showed significant inhibition at a concentration of 10 nM, however, inhibition of O2– production by Ado was significantly less than that in AMP579 at each concentration ranging from 10 nM to 10 µM. The IC50 value for AMP579 (0.09±0.02 µM) on O2– generation was significantly less than that of Ado (3.9±1.1 µM).


Figure 1
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  		Fig. 1 Effects of adenosine (hatched bars) and AMP579 (filled bars) on superoxide radical generation from platelet activating factor (PAF, 5 µM)-stimulated dog polymorphonuclear neutrophils (PMNs, 5x106 cells/ml). Data are presented as nanomoles of superoxide anion produced. CNTL, Control. Values are means±S.E.M. of at least six separate experiments with duplicate determinations. *, P<0.05 vs. PMN plus PAF only group; +, P<0.05 AMP579 vs. adenosine group.

 
3.2 PMN adherence to postischemic coronary artery endothelium
Ischemia–reperfusion significantly increased adherence of unstimulated PMNs to the LAD by 59% in the Control group compared with adherence to the nonischemic LCX (Fig. 2). Although PMN adherence to the LAD in AMPI and AMPIII groups was significantly higher than that of the LCX, continuous infusion of AMP579 in these two groups significantly reduced PMN adherence compared to adherence to the LAD in the Control group. Bolus injection of AMP579 in AMPII and infusion of Ado at reperfusion partially reduced PMN adherence to the LAD. However, adherence in these two groups was significantly greater than that in the AMPI and AMPIII groups, respectively.


Figure 2
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  		Fig. 2 The effects of AMP579 and adenosine on PMN adherence to postischemic coronary artery endothelium. PMNs (4x105/ml). LCX, normal left circumflex coronary artery. Groups: Control (saline control), AMPI (AMP579, 50 µg/kg i.v over 5 min concomitant with 3 µg/kg/min over 2 h), AMPII (AMP579, 50 µg/kg i.v over 5 min), AMPIII (n=7, AMP579, 3 µg/kg/min over 2 h), and Ado (adenosine, 140 µg/kg/min for 2 h). Bar height represents means±S.E.M. of at least 6 separate experiments. *P<0.05 compared with Control group. +P<0.05 AMPI and AMPIII vs. AMPII and Ado groups.

 
3.3 Coronary artery relaxation after ischemia and reperfusion
Coronary vascular responses to acetylcholine (ACh) in isolated rings taken from LAD and LCX are shown in Fig. 3. Untreated ischemia–reperfusion (Control) significantly reduced the endothelium-dependent and muscarinic receptor-mediated vasorelaxation to ACh in the LAD, with a rightward shift of the concentration–response curve and a decrease in maximum relaxation. AMP579 treatment at reperfusion in AMPI and AMPIII groups showed significantly greater vasodilator responses than that in the Control group. Consistent with change in PMN adherence to the LAD, AMP579 (AMPII) and Ado treatment partially restored vascular ring incremental and maximum relaxation compared with LCX; however, there were significant differences between AMPII and Ado versus AMPI and AMPIII, respectively (Fig. 3). Ischemia/reperfusion did not change the responses of the LAD or the LCX to the endothelium-independent smooth muscle vasodilator, nitroprusside (Fig. 4).


Figure 3
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  		Fig. 3 Responses of nonischemic coronary rings (LCX) and ischemic–reperfused coronary rings (LAD) to acetylcholine in Control group (LADC), AMP579 groups (LAD-AMPI, II and III) and Ado group (LAD-Ado). Values are Mean±S.E.M. of at least 15 rings from 6 dogs. *P<0.05 vs. LCX; +P<0.05 vs. LADC.

 

Figure 4
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  		Fig. 4 Responses of nonischemic coronary rings (LCX) and ischemic–reperfused coronary rings (LAD) to nitroprusside in Control group (LADC), AMP579 groups (LAD-AMPI, II and III) and Ado group (LAD-Ado). There were no differences in responses to nitroprusside in five groups Values are Mean±S.E.M. of at least 15 rings from 6 dogs.

 
3.4 Changes in time course of hemodynamics
Hemodynamic data for heart rate (HR), mean aortic pressure (MAP), left ventricular systolic pressure (LVSP), dP/dtmax, left ventricular end-diastolic pressure (LVEDP), and LAD coronary artery blood flow (CBF) in the five groups are shown in Table 1. Coronary occlusion significantly caused an increase in HR. Although MAP, LVSP, and dP/dtmax tended to be less and LVEDP tended to be greater during coronary occlusion in all groups, but none of them reach significant difference compared with baseline values. Infusion of AMP579 and Ado significantly reduced MAP compared with Control group. HR in these treated groups tended to be higher than that in Control group, but it did not reach significance. During the course of reperfusion, HR was still significantly higher than baseline values when other hemodynamic parameters were not significantly changed. In addition, treatment with AMP579 in AMPI and AMPIII groups was associated with a significant increase in LAD blood flow during 2 h of reperfusion.


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  		Table 1 Hemodynamic data measured in control and treatment groupsa

 
3.5 Change in time course of regional contractile function
Paradoxical systolic expansion measured by both systolic shortening (SS) and stroke work (SW) was observed during coronary occlusion in all groups as shown in Table 2. After 2 h of reperfusion, SS and SW in AMPI and AMPIII groups tended to be greater than that in other groups. However, this did not reach statistical significance.


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  		Table 2 Regional contractile function throughout the experimenta

 
3.6 Regional myocardial blood flow after ischemia and reperfusion
Distribution of myocardial blood flow to the nonischemic, ischemic subepicardial (epi-) and subendocadial (endo-) myocardium is shown in Fig. 5. Blood flow in the nonischemic myocardium remained unchanged during the period of coronary occlusion (Fig. 5A) while blood flow in the ischemic epi- and endo-myocardium was reduced by approximately 98% from baseline value in all groups (Fig. 5B and C). There were no group differences in myocardial blood flow during coronary occlusion among the five groups, indicating that any changes in infarct size in AMPI and AMPIII groups was not related to changes in collateral blood flow during ischemia. Release of the coronary snare did not change blood flow in the nonischemic myocardium in the Control group, but resulted in a significantly increased blood flow in ischemic epi- and endo-myocardium in all five groups. Although there was an increase in blood flow in nonischemic and ischemic epi- and endo-myocardium at 15 min of reperfusion in drug-treated groups relative to values in Control group, these values did not reach significant group difference. At 24 h of reperfusion, myocardial blood flow in the ischemic epi-myocardium in AMPI and AMPIII groups was significantly higher than that in Control, AMPII and Ado groups, while no difference between Control versus AMPII and Ado groups was observed (Fig. 5B). In addition, no significant difference in myocardial blood flow was found between Control and drug-treated groups in endo-myocardium at 24 h of reperfusion (Fig. 5C).


Figure 5
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  		Fig. 5 Change in regional myocardial blood flow in nonischemic zone (A), ischemic subepicardial- (B) and subendocardial- (C) myocardium during the course of experiment in the five groups. *, P<0.001 vs. baseline; +, P<0.05 AMPI and AMPIII groups vs. Control, AMPII and adenosine groups.

 
3.7 Myocardial necrotic injury after ischemia and reperfusion
The area placed at risk by coronary occlusion, expressed as a percent of the left ventricular mass (Ar/LV), and the area of necrosis expressed as a percent of the area at risk (An/Ar) are shown in Fig. 6. Ar/LV was comparable among groups. AMP579 administration at reperfusion in AMPI and AMPIII groups significantly reduced An/Ar by 44 and 42%, respectively, compared with Control group. However, a single bolus of AMP579 and intravenous Ado failed to significantly reduce infarct size relative to Control group.


Figure 6
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  		Fig. 6 Area at risk (Ar), expressed as a percentage of left ventricular (LV) mass (Ar/LV) and area of necrosis (An), expressed as a percentage of Ar (An/Ar). Values are group mean±S.E.M. *, P<0.05 vs. Control, AMPII and Ado groups.

 
3.8 Plasma CK activity after ischemia and reperfusion
The plasma CK activity at baseline, during ischemia and reperfusion is shown in Fig. 7. Coronary occlusion only slightly increased CK values, but there were no group differences. CK activity in the Control group was significantly increased at 24 h of reperfusion, reaching a final value of 49±3 I.U./mg protein. AMP579 infusion in AMPI and AMPIII groups showed a lower CK activity relative to other groups at 2 h of reperfusion, but these differences in total CK activity did not reach significance. However, at 24 h of reperfusion, CK activity in AMPI and AMPIII groups was significantly decreased compared to the Control group in agreement with the infarct size data. Although there was a tendency for CK activity to be less in AMPII and Ado groups at 24 h of reperfusion, it did not reach significance.


Figure 7
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  		Fig. 7 Change in plasma creatine kinase (CK) activity during ischemia and reperfusion. Treatment with AMP579 in AMPI and AMPIII groups significantly decreased CK activity at 24 h of reperfusion. Although there was a strong tendency to be less in AMPII and Ado groups on CK activity, they did not reach significance. Values are group mean±S.E.M. *, P<0.001 vs. Control group.

 
3.9 Myocardial tissue edema after ischemia and reperfusion
Tissue edema in nonischemic, ischemic subepicardial and subendocardial myocardium is shown in Table 3. There was no change in tissue edema at 24 h of reperfusion in Control group. In addition, no differences in tissue edema were observed among five groups.


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  		Table 3 Tissue edema for all groupsa

 
3.10 PMN accumulation after ischemia and reperfusion (MPO activity)
Very low MPO activity was detected in the nonischemic zone (Fig. 8). MPO activity was significantly greater in the ischemic zone in the Control group relative to the nonischemic zone. In the AMPI and AMPIII groups, however, MPO activity was significantly decreased by 59 and 55% in the ischemic zone, respectively, suggesting that continuous infusion of AMP579 attenuated PMN accumulation in myocardium. These data were consistent with inhibition of PMN adherence to ischemic coronary artery endothelium in these two groups. There was no significant difference in MPO activity between Control, AMPII and Ado groups. To support a role of PMN in the pathogenesis of infarction and protective effect of AMP579 after ischemia and reperfusion, a linear relationship between MPO activity and infarct size in Control, AMPI and AMPIII groups was plotted. As shown in Fig. 9, MPO activity correlated significantly with the size of infarction at 24 h of reperfusion and the treatment with AMP579 inhibited PMN accumulation with a down-leftward shift of this relationship, suggesting that infarct reduction was correlated with an inhibition of PMN activation during reperfusion.


Figure 8
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  		Fig. 8 Change in tissue myeloperoxidase activity in nonischemic (NI) and ischemic zones in different experimental groups after ischemia and reperfusion. Bar height represents mean±S.E.M. *, P<0.05 vs. Control, AMPII and Ado groups.

 

Figure 9
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  		Fig. 9 Linear relationship between infarct size and myeloperoxidase (MPO) activity after ischemia and reperfusion in Control group (solid circle) and AMP579 groups (AMPI and AMPIII, open circle).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Specific methodology
 3 Results
 4 Discussion
5 Conclusion
 References
 
The present study demonstrated significant infarct reduction and potential mechanisms underlying this cardioprotection by AMP579 during reperfusion. AMP579 inhibited in vitro O2– generation, PMN adherence to coronary endothelium, and augmented the endothelium-dependent vascular relaxation. Continuous infusion of AMP579 at reperfusion increased myocardial blood flow to ischemic myocardium, attenuated PMN accumulation in the ischemic myocardium, and reduced infarct size. Myocardial injury induced by ischemia–reperfusion was not totally reversed by treatment with intravenous adenosine during reperfusion. These results suggest that AMP579 is a potent compound in attenuation of reperfusion injury, and its cardioprotection involves inhibition of PMN-induced vascular and myocardial tissue injury during reperfusion.

4.1 Comparison of AMP579 and adenosine in inhibition of PMN activation and cell–cell interactions in vitro
Comparison of AMP579 and adenosine in inhibition of PMN activation was confirmed by observing attenuation of O2– generation in the present study. At concentrations ranging from 10 nM to 10 µM, AMP579 significantly inhibited O2– generation from activated PMNs, and was about 40-fold more potent than adenosine. The treatment with AMP579 in AMPI and AMPIII groups during reperfusion preserved vascular endothelium from ischemia–reperfusion-induced injury, and therefore, inhibited cell–cell interaction as confirmed by a reduction of PMN adherence to postischemic coronary artery segments. Furthermore, an inhibition of PMN activation by AMP579 in AMPI and AMPIII groups was also demonstrated by attenuation of PMN accumulation in ischemic myocardium. Although adenosine in vitro showed a significant inhibition of PMN activation by reducing O2– generation at a higher concentration (>1 µM), administration of adenosine at reperfusion only partially reversed PMN activation and cell–cell interactions, as confirmed by PMN accumulation in ischemic myocardium and PMN adherence to coronary endothelium. These results suggest that AMP579 is more effective than adenosine in inhibition of PMN-endothelial cell interactions.

4.2 Comparison of continuous infusion of AMP579 versus bolus injection during reperfusion
In the present study, two major regimens were selected for administration of AMP579 during reperfusion, i.e., continuous infusion in AMPI and AMPIII versus bolus injection in AMPII. The protective effect of AMP579 on infarct size in AMPI and AMPIII groups was consistent with previous reports [23–25] in which AMP579 was continuously infused at reperfusion. The estimated in vivo plasma concentration of AMP579 in AMPI and AMPIII was between 10 and 100 nM (based on molecular weight of drug and 80 ml blood volume per kg body weight), suggesting that the dose of 3 µg/kg/min given to achieve this intravascular concentration was sufficient to block O2– generation from activated PMNs according to in vitro data [26]. Although AMP579 given at a 50 µg/kg bolus was sufficient to inhibit PMN activation as estimated from in vivo plasma concentrations, the failure to reduce infarct size by AMP579 in AMPII group suggest that maintaining a constant blood drug level is necessary to protect the heart from ischemia–reperfusion injury. However, absence of direct measurement of AMP579 concentration did not allow us to make a comparison on plasma drug level among groups. In addition, we previously demonstrated a reduction in infarct size by left atrial infusion of adenosine at a dose of 140 µg/kg/min (estimated in vivo concentration between 1 and 10 µM, which show a significant inhibition on PMN activation) for 2 h during reperfusion in a dog model of 1 h ischemia followed by 6 h of reperfusion [17], but its susceptibility to rapid deamination, especially when it is given intravenously, may prevent therapeutic levels from reaching the myocardium. The half life of AMP579, however, is approximately 1 h [21] and this characteristic of AMP579 may ensure that this compound may achieve effective concentrations at the heart, even if it is infused intravenously. We therefore demonstrate from these studies that AMP579 is able to induce cardioprotection when administered only during early reperfusion.

4.3 Prevention of vascular endothelial dysfunction and preservation of myocardial blood flow with AMP579 during reperfusion
Many factors may induce vascular endothelial dysfunction and blood flow defect after ischemia and reperfusion. PMN activation and cell–cell interactions between PMNs and vascular endothelial cells have been suggested as the most important factors for reperfusion-induced damage to the vascular endothelium and myocardial perfusion defects [1,27,28]. Activated and accumulated PMNs after reperfusion mechanically occlude capillaries to increase blood flow resistance, and also release cytotoxic substances (i.e., superoxide radicals) that decrease the vasodilatory response to endogenous vasodilators such as prostacylin and nitric oxide. In studies using either monoclonal anti-PMN CD18 antibody [29] or anti-adhesion molecule ICAM-1 antibody [30,31], attenuated endothelium-dependent vascular relaxation and depressed postischemic capillary perfusion were significantly improved in the model of myocardial ischemia and reperfusion, supporting the role of PMN in induction of endothelial dysfunction and microvascular perfusion defects. In the present study, the concentration-dependent attenuation by AMP579 in O2– generation from PAF-stimulated PMNs, especially in the low concentration range, supports its strong inhibitory effect on PMN activation. The treatment with AMP579 in AMPI and AMPIII groups protected vascular endothelium from ischemia–reperfusion-induced injury, as confirmed by a decreased PMN adherence to postischemic coronary artery segments and improved endothelium-dependent vascular relaxation. In addition, the treatment with AMP579 was also associated with a decreased PMN accumulation in ischemic myocardium. Inhibition of PMN activation and PMN–endothelial cell interactions with AMP579, therefore, may partially explain the protective mechanisms for the improvement of postischemic blood flow defects after ischemia and reperfusion. These data are consistent with the ability of AMP579 to stimulate A2A receptor on PMNs, resulting in inhibiting PMN activation and adhesion [26].

4.4 Failure in protection of regional contractile dysfunction with AMP579 during reperfusion
Regional contractile dysfunction after ischemia and reperfusion is commonly encountered clinically and observed experimentally. PMN activation, oxygen-derived free radical generation and impaired myocardial blood flow have been suggested as major players in this type of injury [32–34]. In the present study, regional contractile function was measured by ultrasonic crystals. At the end of 2 h of reperfusion, systolic shortening and segmental work in the AMPI and AMPIII groups tended to be greater than that in other group, however, this did not reach significance. Although treatment with AMP579 was associated with improvement in postischemic myocardial blood flow, decrease in PMN adherence to coronary endothelium and PMN accumulation in ischemic myocardium, failure in protection of contractile dysfunction with AMP579 suggests that some other factors may also participate in ischemia–reperfusion-induced regional contractile dysfunction. In this regard, reduced ATP availability, calcium overload, dysfunction of the sarcoplasmic reticulum, and interstitial tissue edema have been proposed [35–37].

4.5 Correlation between PMN accumulation and reduction in infarct size by AMP579 during reperfusion
Reduction in infarct size during the early phase of reperfusion (the first 4–6 h) following a brief period of coronary occlusion, either by inhibiting PMN activation and PMN–endothelial cell interactions with monoclonal anti-PMN antibody or depleting PMNs from circulation has been well documented [6,29]. Recent studies from our laboratory and others [11,12,38], however, demonstrated that the degree of PMN accumulation was significantly correlated with the extension of infarction during the first 24 h of reperfusion. Confirmation of this relationship in the present study further suggests that myocardial necrotic injury mediated by PMNs may continue during the late phase of reperfusion [12,20].

We demonstrated previously that intra-atrial administration of adenosine during early reperfusion (6 h) significantly preserved endothelium-dependent vascular relaxation, decreased PMN adherence to ischemic coronary artery endothelium, as well as PMN accumulation in ischemic myocardium, and further reduced infarct size in dog [17]. The present study now provides evidence that adenosine given intravenously was less effective in attenuating PMN-mediated myocardial injury after 24 h of reperfusion. AMP579, however, reduced O2– generation from activated PMNs, eliminated the deleterious effects of activated PMN on endothelium and myocytes, and thereby inhibited the extension of infarct size. A linear relationship between attenuation of PMN accumulation and reduction of infarct size by AMP579 further supports a role of PMN in development of infarction during late reperfusion. The beneficial effects of AMP579 on the ultimate extension of irreversible myocardial injury during reperfusion may relate to its potent inhibitory effect on PMN activation and a long pharmacological half life. It is possible that administration of a higher dose of adenosine may have produced a similar cardioprotective effect although significant hypotension may be difficult to avoid. However, this was not demonstrated in the present study.


   5 Conclusion
Top
 Abstract
 1 Introduction
 2 Specific methodology
 3 Results
 4 Discussion
 5 Conclusion
References
 
In summary, the present study demonstrated that (1) in studies in vitro, AMP579 significantly reduced O2– generation from activated PMN and PMN adherence to coronary artery endothelium, and preserved endothelium-dependent vascular relaxation; (2) in studies in vivo, intravenous administration of AMP579 during reperfusion significantly decreased PMN accumulation, preserved myocardial blood flow and further reduced infarct size, confirmed by a reduction in creatine kinase activity. These studies may provide important insights into the mechanism of action and potential therapeutic efficacy of AMP579 for the attenuation of ischemia–reperfusion injury in patients with thrombolytic therapy, percutaneous transluminal coronary angioplasty or coronary artery bypass graft surgery.

Time for primary review 23 days.


   Acknowledgements
 
The authors are grateful for the assistance of Gail H. Nechtman in preparing the manuscript. This work was supported by a grant from Rhône-Poulenc Rorer Research and Development, Collegeville PA and Carlyle Fraser Heart Center of Emory University School of Medicine, Atlanta, GA.


   References
Top
 Abstract
 1 Introduction
 2 Specific methodology
 3 Results
 4 Discussion
 5 Conclusion
 References
 

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