(Circulation. 1999;100:II-365.)
© 1999 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Platelet-Activating Factor Acetylhydrolase Prevents Myocardial Ischemia-Reperfusion Injury
From the Departments of Surgery and Pathology (E.C.), University of Washington, Seattle, Wash.
Correspondence to Edward D. Verrier, MD, Professor and Chief, Division of Cardiothoracic Surgery, University of Washington, 1959 Pacific Ave NE, Box 356310, Seattle, WA 98195. E-mail verriere{at}ctd.surgery.washington.edu
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Abstract |
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Background—Platelet-activating factor (PAF) is one of the most potent biological mediators of tissue injury. PAF acetylhydrolase (PAF-AH) is a recently isolated naturally occurring enzyme that hydrolyzes PAF and renders it inactive. We hypothesize that inhibition of PAF with PAF-AH will reduce myocardial ischemia-reperfusion (I/R) injury in vivo.
Methods and Results—The coronary ligation model was used in New Zealand white rabbits. The large branch of the marginal coronary artery was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given either 2 mg/kg of vehicle or of PAF-AH. At the completion of 120 minutes of reperfusion, percentage of necrosis, degree of neutrophil infiltration, and measurements of regional contractility were assessed. Data are expressed as the mean±SEM and compared by Student’s t test or Mann-Whitney ANOVA. Both groups of animals showed an equivalent area at risk; however, 46.7±11% was necrotic in the animal treated with vehicle. In contrast, 20.9±7.0% was necrotic in the animals treated with PAF-AH (P<0.05). Systolic shortening and wall thickness were significantly greater in those animals treated with PAF-AH at 15, 30, 60, and 120 minutes of reperfusion (P<0.05). Quantification of neutrophil infiltration showed a 62% reduction in the PAF-AH treated animals compared with those treated with vehicle alone.
Conclusions—PAF-AH is a potent cardioprotective agent in an in vivo model of I/R injury.
Key Words: endothelium-derived factors • ischemia • reperfusion • myocardial infarction
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Introduction |
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This year, as many as 1.5 million Americans will have a new or recurrent myocardial infarction, and as a direct result, one third of these people will die.1 Although several types of interventions exist to treat this disorder, including PTCA and CABG, significant potential still exists for morbidity and mortality in the form of myocardial ischemia-reperfusion (I/R) injury.
When the heart is rendered ischemic, a reduction occurs in the natural antioxidant defenses of the cells. At reperfusion, an accumulation of reactive oxygen intermediates results in a change in the redox potential of the cell, known as oxidative stress. Oxidative stress initiates a host inflammatory response characterized by widespread neutrophil adhesion and activation, initiation of coagulation, and alteration in vascular permeability.
Platelet-activating factor (PAF) is a potent biological mediator of inflammation and has been shown to participate significantly in I/R injury.2 Under normal physiological conditions, PAF is minimally expressed; however, under conditions of oxidative stress, as occurs in I/R injury, PAF is released by neutrophils and monocytes and is expressed on the outer leaflet of endothelial cells.3 Once expressed, PAF results in several diverse biological activities, including neutrophil activation and chemotaxis, alterations in vascular permeability, platelet activation, and negative inotropy, all of which may contribute to the clinical manifestations of I/R injury.4 5
The tissues, blood cells, and plasma of the body all have enzymes that catalyze the hydrolysis of PAF. This self-protective mechanism limits the damaging effect of PAF.6 Studies with newly available human recombinant PAF-acetylhydrolase (AH) have shown that it reduces edema, prevents asthma-related symptoms, and protects animals from septicemia.7 8 The protective effects of PAF-AH have never been studied under conditions of myocardial oxidative stress. The purpose of the present study was to test the hypothesis that PAF-AH reduces myocardial I/R injury.
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Methods |
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In this model, adult New Zealand White rabbits weighing 3 to 4 kg each were used in research protocols approved by the Animal Care Committee of the University of Washington, Seattle. All animals received humane care according to the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23).
Reagents
PAF-AH and the vehicle in which it was dissolved were acquired
from ICOS Corp (Bothell, Wash). Both compounds, at a dose of 2
mg/kg, were given intravenously through the marginal ear
vein 15 minutes before reperfusion. Twelve animals received PAF-AH: 6
were used for infarct data and 6 for histology. Twelve animals received
vehicle: 6 were used for infarct data and 6 for histology. To ensure
that recombinant PAF-AH alone had no effect on study end points, 3
animals received PAF-AH without ischemia; all were used for
regional contractility data and histology.
Surgical Procedure
Rabbits were anesthetized with an initial
intramuscular injection of ketamine (35 mg/kg) and xylazine (5
mg/kg). The rabbits were endotracheally intubated and maintained on
inhaled halothane (1% to 2%) anesthesia in 100%
O2 using a small-animal respirator. A 20-gauge
flexible catheter was placed in the left carotid artery to measure
heart rate (HR) and mean arterial pressure and to collect
blood samples. The heart was exposed via median sternotomy. A 5F Millar
catheter was placed through a small pursed-string incision in the left
ventricle to allow estimation of the left ventricular peak
systolic pressure, left ventricular end
diastolic pressure, and maximum and minimum change in left
ventricular pressure (dP/dt). One pair of sonometric
piezoelectric crystals was placed on the surface of the heart to assess
regional wall motion abnormalities via segmental shortening (SS) in the
area at risk. Another crystal was tunneled tangentially to the
subendocardium, with a second crystal sutured to the epicardium for
measurements of LV wall thickness (WT) (Sonometrics, Inc). The
hemodynamic trace acquired from the Millar catheter
allowed SS and WT measurements to be correlated with
end-diastolic and end-systolic time points. A 4.0
Vicryl suture was passed twice around a large anterolateral branch of
the left main coronary artery that supplied to most of the left
ventricle, and the ends of the suture passed through a small length of
polyethylene tubing to form a snare. After a 20- to 30-minute
stabilization period, baseline values were recorded. Regional
myocardial ischemia was produced by reversibly occluding the
artery for 45 minutes, followed by 120 minutes of reperfusion. Fifteen
minutes before reperfusion, the rabbits received 2.0 mg/kg of PAF-AH or
vehicle intravenously. Heart rate, mean
arterial pressure, left ventricular
end-systolic pressure, left ventricular
end-diastolic pressure, ±dP/dt, WT, and SS were
recorded at baseline; 30 minutes of ischemia; 45 minutes
ischemia; and 15, 30, 60, and 120 minutes after reperfusion.
Animals that received PAF-AH alone had the same measurements
recorded at baseline and 15, 30, 60, and 120 minutes after drug
administration. After 120 minutes of reperfusion, all animals were
euthanatized with an intravenous bolus of concentrated
pentobarbital, and the myocardial tissue was taken and processed for
calculation of infarct size or histologic analysis.
Determination of Infarct Size
At the completion of the 120-minute reperfusion period, the
coronary artery was reoccluded and 6 mL of 20% Evans blue dye
(Sigma Chemical Co) was injected into the left atrium and allowed to
circulate and stain all perfused tissue. The area supplied by the
ligated vessel remained unstained, which demarcated the
myocardium at risk of infarction. After the heart was
arrested with pentobarbital, it was rapidly excised. The left ventricle
was isolated from the rest of the heart, weighted, and then cut into
2-mm-thick transverse slices. The normal myocardium
(stained blue) was separated from the area at risk (unstained). The
area at risk was placed in a 37°C solution of 1%
triphenyltetrazolium chloride (TTC) for 30
minutes. TTC stains the viable tissue brick red, which leaves the
necrotic zone pale. The TTC red (noninfarcted) tissue is separated from
the TTC pale (necrotic) tissue, and each area is weighed. The left
ventricular area at risk was calculated as the sum of the
noninfarcted and necrotic tissue weight of the tissue perfused by the
occluded vessel divided by the weight of the left ventricle, expressed
as a percentage. The left ventricular area of necrosis was
calculated as the weight of necrotic tissue divided by the weight of
the left ventricle, expressed as a percentage. The infarct size was
calculated by dividing the weight of the TTC pale tissue by the weight
of the total area at risk (left ventricular area of
necrosis/left ventricular area at risk).
Determination of Regional Contractility
End-diastolic length, end-systolic length,
and WT were determined from the strip-chart recording by an
observer blinded to the study design. Percentage SS of baseline was
calculated by the following equation:
100x[(EDL-ESL)/EDSB]=%SS, where EDL
indicates end-diastolic length; ESL, end-systolic
length; and EDSB, end-diastolic length–end
systolic length at baseline. Percentage WT change was
calculated by the following equation:
100x(ESW-EDW/EDSB)=%Change in WT, where ESW
indicates end-systolic width; EDW, end-diastolic
width; and EDSB, end-systolic width minus
end-diastolic width at baseline.
Determination of Neutrophil Infiltration
Neutrophil accumulation in the heart was quantified in the
following manner: A tissue block measuring 
1.0x1.0x0.5 cm was
randomly selected from each rabbit heart. The blocks were dehydrated
and embedded in paraffin, and 3-mm sections were placed on glass
slides. The slides were stained with hematoxylin and eosin. A
pathologist blinded to the study protocol counted the number of
neutrophils in 10 randomly selected fields (2200
µm2).
Statistical Analysis
The data analysis was performed using SPSS for Windows,
version 6.1. For each of the treatment groups, several
parameters (expressed as continuous variables) were
studied at the indicated time points. To determine whether a
statistically significant change had occurred compared with the
baseline value, the parameters were compared with baseline
by use of paired t tests. For intergroup comparisons, the
mean change (from baseline) for each of these parameters
was analyzed at the specific time points using ANOVA. The
Mann-Whitney test was used for post hoc comparisons. A probability
value of <0.05 was considered statistically significant.
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Results |
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PAF-AH Reduces Infarct Size
The percentages of areas at risk for infarction were similar in both groups (vehicle, 44.3 ± 4.3%; PAF-AH, 46.73± 2.7%) as depicted in Figure 1
. However, infarct
size was markedly reduced in the group treated with PAF-AH
(20.97±3.3%) compared with the vehicle-treated group (45.72±6.5%),
P<0.05.
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PAF-AH Reduces Regional Myocardial Wall Motion
Abnormalities
Wall Thickness
A similar reduction in WT after coronary occlusion
occurred in both vehicle and PAF-AH groups (34% vehicle versus 49%
PAF-AH group). By 120 minutes after reperfusion, the vehicle-treated
animals had recovered to an average of only 45% of the baseline value,
whereas the PAF-AH–treated group had recovered to an average of 85%
of the baseline value (Figure 2). Animals
that received PAF-AH alone maintained nearly 100% of the baseline
value throughout the entire sampling period (data not shown).
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Systolic Shortening
Coronary occlusion similarly reduced
systolic shortening to 50% and 75% of baseline values in the
vehicle and PAF-AH treated groups, respectively. However, after 120
minutes of reperfusion, the vehicle group had recovered only 54.5% of
its baseline regional contractile function, whereas the PAF-AH group
recovered to 103% of its baseline value (Figure 2
). Animals
that received PAF-AH alone maintained 100% of the baseline value
throughout the sampling period (data not shown).
PAF-AH Reduces Neutrophil Infiltration
Histologic examination of myocardial tissue after I/R in the area
at risk for infarction, revealed marked
endothelial-leukocyte adhesion and
transendothelial migration in those animals receiving
vehicle alone. In PAF-AH-treated animals, in contrast, neutrophil
adherence was markedly reduced. Animals treated with vehicle alone had
157 polymorphonuclear neutrophils (PMNs) per high-powered
field compared with 75 PMNs per high-powered field in PAF-AH–treated
animals. Animals that received PAF-AH in the absence of
ischemia averaged only 1 PMN per high-powered field (data not
shown). These results suggest that PAF-AH inhibited
neutrophil–endothelial cell interactions in vivo,
although the difference in neutrophil accumulation was not significant
(Figures 3 and 4). The enzyme alone appears to have no
effect on neutrophil activation and migration, which correlates with
unpublished data in the lung, kidney, and pancreas (Brian Schimpf, PhD,
personal communication with ICOS, 1999).
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Discussion |
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These results suggest that PAF plays a role in the regional cardiac dysfunction associated with I/R injury. The administration of its natural inhibitor, PAF-AH, markedly reduces this injury. Specifically, PAF-AH reduces infarct size and enhances regional ventricular functional recovery after I/R.
Several mechanisms exist by which PAF-AH may reduce I/R injury. One of the cardioprotective features of PAF-AH may stem from its ability to modulate leukocyte-endothelium interactions. The endothelial cell, when exposed to conditions of oxidative stress, synthesizes PAF within minutes and transports it to the plasma membrane, at which it is expressed on the outer leaflet of the plasma membrane. Once expressed, PAF serves as both a chemoattractant and activator of neutrophils, which results in neutrophil-endothelial adherence, superoxide anion release, and degranulation.2 3 Furthermore, PAF may also result in vasoconstriction,9 alteration in vascular permeability,10 and negative ionotropy11 in the setting of I/R. Any of these biological activities could explain the beneficial effect that exogenous administration of PAF-AH has in our model.
Much of the evidence implicating the role of PAF in I/R injury has been acquired through the use of PAF antagonists. PAF antagonists used in in vivo models of I/R have proved to be cardioprotective, as indicated in functional measurements such as pressure-volume loops in swine,11 dP/dt in sheep,12 and systolic developed pressure in rabbits.13 Their use has also resulted in reduction of infarct size in the reperfused myocardium and inhibition of reperfusion-induced coronary artery vasoconstriction.14 Although these antagonists have proven to be a valuable research tool for elucidation of the role of PAF in I/R injury, they have also proven to be problematic when used clinically. Their detrimental effects include PAF agonist activities at high concentrations, antagonism of ADP and collagen production, significant hemolysis, and vascular damage.2
In 1995, the cDNA encoding the plasma form of PAF-AH was cloned.8 The primary structure of plasma PAF-AH is unique. Its active site contains a catalytic triad found in many phospholipases that contains a serine, an acidic (usually aspartate), and a histidine residue. This catalytic triad hydrolyzes the acyl group at the second position of glycerol moieties. However, PAF-AH, unlike other phospholipases, has marked selectivity for phospholipids with short acyl chains at the sn-2 positions.7 Thus, normal membrane phospholipids are protected from hydrolysis by an intrinsic property of the enzyme. This same selective activity highlights its ability to be the most specific inhibitor of PAF known. As a result, this recombinant enzyme lacks the detrimental, nonselective activity of other PAF antagonists.
Early experience with PAF-AH in clinical trials has shown it to be a safe and efficacious means to treat inflammatory-mediated pulmonary dysfunction. Additionally, an increase in PAF levels has been documented during cardiac surgery with cardiopulmonary bypass. This finding did not correlate with a difference in serum PAF-AH activity.15 Therefore, exogenous administration of PAF-AH may prove to be a viable clinical means to reduce the detrimental activity of excessive PAF, which could result in a reduction in both myocardial injury and the generalized inflammatory consequences of cardiopulmonary bypass.
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Acknowledgments |
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This work was funded in part by the Thoracic Surgery Foundation for Research and Education Fellowship (Dr Morgan and Dr Boyle); National Institutes of Health grants GM-46662 and T32-GM-07037 (Dr Pohlman); and the ICOS Corp. We thank Robert Thomas; Christine Rothine; Angela Farr; Ellen Collins; A. Travis Nease; and Jeanette Griscavage-Ennis, PhD, for their assistance in the laboratory and Michael Caps, MD, MPH, for his review of the statistical comparisons.
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