Cardiovascular Research 47 (2000) 294–305
www.elsevier.com / locate / cardiores
www.elsevier.nl / locate / cardiores
Comparative study of AMP579 and adenosine in inhibition of neutrophilmediated vascular and myocardial injury during 24 h of reperfusion
a
a
a,
b
a
Jason M. Budde , Daniel A. Velez , Zhi-Qing Zhao *, Kenneth L. Clark , Cullen D. Morris ,
Satoshi Muraki a , Robert A. Guyton a , Jakob Vinten-Johansen a
a
Cardiothoracic Research Laboratory, The Carlyle Fraser Heart Center /Crawford Long Hospital, Emory University School of Medicine,
550 Peachtree St., NE, Atlanta, GA 30365 -2225, USA
b
ˆ
Rhone-Poulenc
Rorer Research and Development, Collegeville, PA 19426, USA
Received 21 February 2000; accepted 25 April 2000
Abstract
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 (n58, saline control), AMPI (n57, AMP579, 50 mg / kg i.v. bolus followed by 3 mg / kg / min for 2 h), AMPII
(n57, AMP579, 50 mg / kg i.v. bolus), AMPIII (n57, AMP579, 3 mg / kg / min i.v. for 2 h), and Ado (n57, adenosine, 140 mg / kg / min i.v.
6
for 2 h). Results: AMP579 in vitro directly inhibited superoxide radical (O 2
PMNs) from PMNs dose2 ) generation (nM / 5310
2
dependently (from 1761* at 10 nM to 260.2* at 10 mM vs. activated 3062). However, inhibition of O 2 generation by Ado at each
concentration was significantly less than for AMP579. The IC 50 value for AMP579 (0.0960.02 mM) on O 22 generation was significantly
less than that of Ado (3.961.1 mM). Adherence of unstimulated PMN to postischemic coronary artery endothelium (PMNs / mm 2 ) was
attenuated in AMPI and AMPIII vs. Control (6063* and 5863* vs. Control 11064), while Ado partially attenuated PMN adherence
(9863*). 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 (0.860.1* and 0.760.1* vs. Control 0.360.04). Infarct size (IS, TTC staining) in AMPI and AMPIII was
significantly reduced from 3863% in Control to 2164%* and 2263%*, respectively, confirmed by lower plasma creatine kinase activity
(I.U. / g protein) in these two groups (2766* and 3262* vs. 4963). Cardiac myeloperoxidase activity (MPO, Abs / min) in the AAR was
significantly reduced in AMPI and AMPIII vs. Control (36611* and 35610* vs. 89610). 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.  2000 Elsevier Science B.V. All rights reserved.
Keywords: Adenosine; Free radicals; Ischemia; Leucocytes
1. Introduction
Abundant evidence from both experimental animals and
clinical observations indicate that reperfusion-induced
*Corresponding author. Tel.: 11-404-686-2511; fax: 11-404-6864888.
E-mail address: [email protected] (Z.-Q. Zhao).
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
Time for primary review 23 days.
0008-6363 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 00 )00115-2
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
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 PMNmediated 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 intraatrial 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-2thienyl) 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 A 1 (Ki 55 nM) and A 2A (Ki 556 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 closedchest ischemia–reperfusion model.
295
2. Specific methodology
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 (n58, saline control), AMPI (n57, AMP579, 50
mg / kg i.v. bolus followed by 3 mg / kg / min for 2 h),
AMPII (n57, AMP579, 50 mg / kg i.v. bolus), AMPIII
(n57, AMP579, 3 mg / kg / min for 2 h), and Ado (n57,
adenosine, 140 mg / 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
296
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
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 48C 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 378C 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 Odianisodine dihydrochloride and H 2 O 2 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 nonischemic, ischemic subepicardial and subendocardial
myocardium were separated. Myocardial edema formation
was quantified by the calculation of myocardial water
content: % H 2 O5[12(dry weight / wet weight)]3100.
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 FicollHypaque 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 ( O 2
2 ) production
O2
production
by
PMNs
was
determined
by measuring
2
the O 2
dismutase-inhibitable
reduction
of
ferricytochrome
2
C to ferrocytochrome C using a Spectra Max 250 microplate reader spectrophotometer. PMNs (5310 6 / 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 (4310 5 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 378C. After stabilization, the coronary
rings were subsequently preconstricted with thromboxane
A 2 –mimetic U46619 (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
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
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-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
3.1. Generation of O 2
2 from activated PMNs
Inhibition of O 2
2 generation from PMNs by AMP579
and Ado is shown in Fig. 1. AMP579 inhibited O 22
production in a concentration-dependent manner. AMP579
showed significant inhibition at a concentration of 10 nM,
however, inhibition of O 2
2 production by Ado was significantly less than that in AMP579 at each concentration
ranging from 10 nM to 10 mM. The IC 50 value for
Fig. 1. Effects of adenosine (hatched bars) and AMP579 (filled bars) on
superoxide radical generation from platelet activating factor (PAF, 5
mM)-stimulated dog polymorphonuclear neutrophils (PMNs, 5310 6
cells / ml). Data are presented as nanomoles of superoxide anion produced. CNTL, Control. Values are means6S.E.M. of at least six separate
experiments with duplicate determinations. *, P,0.05 vs. PMN plus PAF
only group; 1, P,0.05 AMP579 vs. adenosine group.
297
AMP579 (0.0960.02 mM) on O 2
2 generation was significantly less than that of Ado (3.961.1 mM).
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.
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
Fig. 2. The effects of AMP579 and adenosine on PMN adherence to
postischemic coronary artery endothelium. PMNs (4310 5 / ml). LCX,
normal left circumflex coronary artery. Groups: Control (saline control),
AMPI (AMP579, 50 mg / kg i.v over 5 min concomitant with 3 mg / kg /
min over 2 h), AMPII (AMP579, 50 mg / kg i.v over 5 min), AMPIII
(n57, AMP579, 3 mg / kg / min over 2 h), and Ado (adenosine, 140
mg / kg / min for 2 h). Bar height represents means6S.E.M. of at least 6
separate experiments. *P,0.05 compared with Control group. 1P,0.05
AMPI and AMPIII vs. AMPII and Ado groups.
298
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
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 Mean6S.E.M. of at least 15 rings from 6 dogs. *P,0.05 vs. LCX;
1P,0.05 vs. LADC.
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).
3.4. Changes in time course of hemodynamics
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 Mean6S.E.M. of at least 15 rings from 6 dogs.
3.5. Change in time course of regional contractile
function
Hemodynamic data for heart rate (HR), mean aortic
pressure (MAP), left ventricular systolic pressure (LVSP),
dP/ dt max , 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/ dt max 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.
Paradoxical systolic expansion measured by both
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
299
Table 1
Hemodynamic data measured in control and treatment groups a
Time
Groups
Parameters
HR
MAP
LVSP
LVEDP
1dP/ dt
2dP/ dt
CBF
Baseline
Control
Ado
AMPI
AMPII
AMPIII
8466
84610
8364
97610
89612
9566
8863
9368
8867
9368
10666
10763
10464
9162
10967
8.761.3
861.7
10.760.9
6.861.3
11.361.6
16286248
17196218
1633673
1645693
20276131
21443696
213656105
21474671
21353671
216046141
12.662
10.262.2
11.962.5
12.661.6
9.161.3
Ischemia
Control
Ado
AMPI
AMPII
AMPIII
11067*
12768*
10166*
134616*
10468*
9166
9264
8464
8063
8667
10266
10663
9665
9163
10066
11.361.5
11.961.6
1361.4
8.561.1
13.262.2
14996137
13626175
14086195
1335672
1531689
213656102
213656106
21276694
21079673
214196121
2
–
–
–
–
R0h
Control
Ado
AMPI
AMPII
AMPIII
11267*
13469*
12766*
143614*
122614*
8967
7764*
6863*
6864*
7066*
10365
9263
8264
8563*
8765
10.361.5
9.961.6
8.461.7
6.761
11.362.1
14396132
12626175
13356153
1398671
14796104
21305699
212456106
210896167
2956675
29266121
–
–
–
–
–
R2h
Control
Ado
AMPI
AMPII
AMPIII
118610*
129610*
13466*
11069*
12766*
8363
8263
8164
7662
7465
9864
10064
9865
8162
8865
7.661.5
8.561.8
10.561.8
7.561.5
8.261.4
15936137
13916191
1644663
1384686
1636678
213656103
213656107
21493698
211546103
21396681
14.662.7
17.164.8
28.467.4*
16.862.5
29.968.6*
R24th
Control
Ado
AMPI
AMPII
AMPIII
13263*
13965*
13166*
12565*
13669*
7465
8965
8566
7563
8069
8965
10263
9967
8667
9267
8.261
9.161.2
9.661.6
7.560.9
7.460.9
1216124
12706167*
13966105
1350675
1305689*
213656104
213656108
214256106
212746108
213236119
6.361
11.362.7
1362.1
11.163.1
10.561.9
a
HR, heart rate (beats / min); MAP, mean aortic pressure (mmHg); LVSP, left ventricular peak systolic pressure (mmHg); LVEDP, left ventricular
end-diastolic pressure (mmHg); positive dP/ dt (mmHg / s); negative dP/ dt (mmHg / s); CBF, mean coronary (LAD) blood flow (ml / min). R0h, 5 min after
drug infusion and before reperfusion. R2h and 24th, 2 and 4 h of reperfusion. Control (saline control), Ado (adenosine, 140 mg / kg / min infusion for 2 h),
AMPI (AMP579, 50 mg / kg i.v. bolus over 5 min followed by 3 mg / kg / min infusion over 2 h), AMPII (AMP579, 50 mg / kg i.v. bolus over 5 min), AMPIII
(AMP579, 3 mg / kg / min infusion over 2 h). Values are expressed as means1S.E.M. *P,0.05 versus baseline value.
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.
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 endomyocardium 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).
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
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
300
Table 2
Regional contractile function throughout the experiment a
Time
Groups
Parameters
EDL (mm)
ESL (mm)
SS (%)
SW (mmHg / min)
Baseline
Control
Ado
AMPI
AMPII
AMPIII
13.261.0
14.562.2
12.861.1
13.560.7
13.760.7
11.260.8
11.861.9
8.760.9
9.260.5
10.560.7
15.261.7
19.262.6
19.763.1
26.162.0
20.462.4
180627
267636
176632
183627
216636
Ischemia
Control
Ado
AMPI
AMPII
AMPIII
14.861.0
16.461.6
12.660.8
15.460.6
14.960.7
15.461.0*
16.761.8*
12.861.1*
16.060.8*
15.460.8*
24.261.0*
21.962.0*
23.263.0*
23.46.7*
23.561.7*
1662*
20620*
34625*
2165*
32613*
R0h
Control
Ado
AMPI
AMPII
AMPIII
14.861.0
16.461.6
11.060.8
14.060.6
14.860.7
15.461.0*
16.761.8*
10.861.1*
14.460.8*
15.160.8*
24.261.0*
21.962.0*
20.363.0*
23.263.0*
22.061.7*
1662*
20620*
38625*
2665*
37613*
R2h
Control
Ado
AMPI
AMPII
AMPIII
13.861.0
14.861.5
11.460.7
12.560.9
12.760.8
13.861.0*
14.861.6*
10.461.0*
12.960.7*
11.560.8*
0.1161.3*
21.362.4*
3.363.1*
22.161.4*
4.762.4*
26613*
25614*
65617*
1866*
65618*
R24th
Control
Ado
AMPI
AMPII
AMPIII
14.760.8
14.161.7
10.261.4
12.361.0
12.261.1
14.160.8*
14.161.6*
10.061.1*
12.361.0*
11.661.2
20.3161.6*
0.660.9*
2.361.7*
0.460.3*
2.862.9*
2168*
762*
17619*
10610*
17618*
a
EDL, end-diastolic length; ESL, end-systolic length. SS, systolic shortening; SW, segmental work. R0h, 5 min after drug infusion and before
reperfusion; R2h, R4h, 2,4 h afters reperfusion. All values are expressed as mean1S.E.M. *, P,0.05 versus baseline value.
(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.
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.
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 4963 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.
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
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
301
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 mean6S.E.M. *, P,0.05 vs. Control,
AMPII and Ado groups.
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; 1, P,0.05 AMPI and AMPIII groups vs. Control, AMPII and
adenosine groups.
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.
4. Discussion
The present study demonstrated significant infarct reduction and potential mechanisms underlying this cardioprotection by AMP579 during reperfusion. AMP579
inhibited in vitro O 2
2 generation, PMN adherence to coronary endothelium, and augmented the endothelium-
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 mean6S.E.M.
*, P,0.001 vs. Control group.
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 reperfuTable 3
Tissue edema for all groups a
Group
NIZ
EPIZ
ENDZ
Control
Ado
AMPI
AMPII
AMPIII
78.4
77.9
77.1
75.9
75.4
80.1
79.2
78.9
77.2
78.1
79.1
80.5
79.7
77.9
79.4
a
NIZ, nonischemic zone; EPIZ, subepicardial zone; ENDZ, subendocardial zone. All values are expressed as percent change.
302
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
dium. Although adenosine in vitro showed a significant
inhibition of PMN activation by reducing O 2
2 generation at
a higher concentration (.1 mM), 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 PMNendothelial cell interactions.
Fig. 8. Change in tissue myeloperoxidase activity in nonischemic (NI)
and ischemic zones in different experimental groups after ischemia and
reperfusion. Bar height represents mean6S.E.M. *, P,0.05 vs. Control,
AMPII and Ado groups.
sion 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
O 22 generation in the present study. At concentrations
ranging from 10 nM to 10 mM, AMP579 significantly
inhibited O 2
2 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 myocar-
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).
4.2. 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 mg / kg / min given to achieve
this intravascular concentration was sufficient to block O 2
2
generation from activated PMNs according to in vitro data
[26]. Although AMP579 given at a 50 mg / 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 mg / kg / min (estimated in vivo concentration between 1 and 10 mM, 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 dysfunc-
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
tion 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 concentrationdependent attenuation by AMP579 in O 2
2 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 A 2A 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 dys-
303
function. 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 O 2
2 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
In summary, the present study demonstrated that (1) in
studies in vitro, AMP579 significantly reduced O 2
2 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
304
J.M. Budde et al. / Cardiovascular Research 47 (2000) 294 – 305
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.
[14]
[15]
[16]
Acknowledgements
The authors are grateful for the assistance of Gail H.
Nechtman in preparing the manuscript. This work was
ˆ
supported by a grant from Rhone-Poulenc
Rorer Research
and Development, Collegeville PA and Carlyle Fraser
Heart Center of Emory University School of Medicine,
Atlanta, GA.
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