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1 Department of Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3392; and 2 Discovery Research, Pharmacia and Upjohn, Kalamazoo, Michigan 49001
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We investigated
in vivo coronary P-selectin expression and its pathophysiological
consequences in a murine model of myocardial ischemia-reperfusion (MI/R) using wild-type and P-selectin
deficient (
/) mice. Coronary P-selectin expression
[µg monoclonal antibody (MAb)/g tissue] was measured
using a radiolabeled MAb method after 30 min of myocardial
ischemia and 20 min of reperfusion. P-selectin expression in
wild-type mice was significantly (P < 0.01) elevated in the ischemic zone (0.070 ± 0.010) compared
with the nonischemic zone (0.037 ± 0.008). Myocardial P-selectin
expression was nearly undetectable in P-selectin / mice
after MI/R. Furthermore, myocardial infarct size (% of area at risk)
after 30 min of myocardial ischemia and 120 min of reperfusion
was 42.5 ± 4.4 in wild-type mice and 24.4 ± 4.0 in P-selectin
/ mice (P < 0.05). In
additional experiments of prolonged myocardial ischemia (60 min) and reperfusion (120 min), myocardial infarct size was similar in
P-selectin / mice and wild-type mice. Our results clearly
demonstrate the involvement of coronary P-selectin in the development
of myocardial infarction after MI/R.
neutrophil; adhesion molecules; infarction; mouse; monoclonal antibody
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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PREVIOUS STUDIES (19-21) showed an association between leukocyte-endothelial cell adhesion molecule (ECAM) expression and enhanced neutrophil (PMN)-endothelial cell interactions. Furthermore, it has been demonstrated that PMN activation causes inflammation (26), endothelial cell dysfunction (30), and, ultimately, cellular necrosis (4). Accordingly, the level of leukocyte-ECAM expression is an important factor in the involvement of PMNs in tissue destruction. This varied involvement of PMNs warrants investigation into their specific roles in the evolution of tissue damage and organ dysfunction.
Myocardial ischemia-reperfusion (MI/R) injury is appreciated as a potent stimulus for tissue destruction and possible cardiac failure (4, 11). Much of this injury is believed to result from intensified PMN-endothelial cell affinity via enhanced ECAM expression subsequent to MI/R. In particular, the early expression of P-selectin may have a profound impact on PMN-mediated injury after MI/R (23). Although there are a variety of ECAMs involved in PMN-mediated injury, the upregulation of P-selectin is an early and necessary step in PMN sequestration. It has previously been demonstrated (9, 23) that in vivo leukocyte tethering to the endothelium is highly dependent on endothelial cell P-selectin expression. Furthermore, it has also been demonstrated that administration of monoclonal antibodies (MAbs) (36) or carbohydrate ligands (25) directed against P-selectin markedly reduces myocardial reperfusion injury in animal models of MI/R. These studies suggest a pivotal role for P-selectin in MI/R injury.
In the present study, we used a novel dual-radiolabeled MAb technique to quantify regional P-selectin expression in vivo in a murine model of MI/R. We also used mice that were genetically targeted to be deficient in P-selectin to compare the extent of myocardial injury in P-selectin-deficient and wild-type mice after various MI/R protocols. Clarifying the level of coronary P-selectin expression and the pathological consequences of P-selectin upregulation is an important step in understanding the mechanisms that underlie MI/R injury. Furthermore, this understanding may contribute to pharmacological therapies for myocardial infarction in humans.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Transgenic mice.
The P-selectin-deficient (/) mice used in the present
study were developed as described by Bullard et al. (6) and were kindly
provided by Pharmacia and Upjohn (Kalamazoo, MI). Age- and
weight-matched wild-type mice (C57BL/6) were purchased from Jackson
Laboratories (Bar Harbor, ME). All experimental procedures complied
with the Guide for the Care and Use of Laboratory
Animals [DHHS Publication No. (NIH) 86-23,
revised 1985. Bethesda, MD 20892] and with federal and state
regulations. All experimental procedures were approved by the Louisiana
State University Medical Center Animal Care and Use Committee.
Surgical procedures. Animals were initially anesthetized with pentobarbital sodium (100 mg/kg ip) before any surgical procedure. Anesthesia was maintained via supplemental doses of pentobarbital sodium (30 mg/kg ip) as needed. Mice were secured to the operating table by taping the extremities. A 4-0 silk ligature was placed behind the upper incisors and pulled tautly to extend the neck. A midline incision was made from the xiphoid process to the submentum. The salivary glands were separated from the midline to allow access to the trachea. A tracheotomy was then performed, and a section of polyethylene-90 tubing was inserted into the animal's trachea and connected via a loose junction to a Harvard respirator (model 683 rodent respirator, Harvard Apparatus). The tidal volume of the respirator was set at 1.8 ml/min, with the rate set at 120 strokes/min, and was supplemented with 100% oxygen. The right carotid artery was then cannulated with polyethylene-10 tubing to monitor mean arterial pressure. The arterial cannula was connected to a blood pressure transducer and BP-1 (World Precision Instruments) blood pressure monitor.
After an equilibration period of 20 min, a thoracotomy was performed. Using electrocautery (model 100, Geiger Instrument), an incision was made to the left of the sternum. Retraction stitches (4-0 silk) were used on each side of the thoracotomy to aid in visualization of the heart. The pericardial sac was then removed. Ligation of the left anterior descending coronary artery (LAD) was performed using a 7-0 silk suture attached to a BV-1 needle (Ethicon). A small piece of polyethylene tubing was used to secure the ligature without damaging the artery. The retraction sutures were removed, and the chest wall was approximated and covered with Parafilm to prevent desiccation.MI/R infarct protocols.
A diagram of the MI/R protocol is presented in Fig.
1. One group of wild-type
(n = 6) and P-selectin /
(n = 10) mice was subjected to 30 min
of LAD occlusion and 120 min of reperfusion. In subsequent studies, a
second group of wild-type (n = 7) and P-selectin / (n = 7)
mice was subjected to 60 min of LAD occlusion and 120 min of
reperfusion.
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Determination of area at risk and infarct size. At the conclusion of the 2-h period of reperfusion, the LAD was religated with 7-0 silk suture. Evans blue dye (1.5 ml, 1.0%; Sigma) was retrogradely injected into the carotid artery catheter to delineate the in vivo area at risk (AAR).
At the end of the protocol, the heart was excised and fixed in 1.5% solution of SeaPlaque agarose gel (FMC BioProducts). After the gel solidified, the heart was sectioned perpendicular to the long axis in 1-mm portions using a McIlwain tissue chopper (Brinkmann Instruments). The 1-mm sections were placed in individual wells of a six-well cell culture plate (Cell Wells; Corning Glass Works) with the basal side exposed. Each slice was then counterstained with 3.0 ml of 1.0% 2,3,5-triphenyltetrazolium chloride (Sigma) solution for 5 min at 37°C. The right ventricle was excised, and each slice was weighed and visualized under an Olympus SZ4045 (Olympus America) dissecting microscope equipped with a Sony charge-coupled device Iris color video camera (Sony Electronics). The left ventricular (LV) area, AAR, and area of infarction for each slice were then determined by computer planimetry using NIH Image (version 1.57) software. The size of the myocardial infarction was determined by the following previously described equation (29): weight of infarction = (A1 × Wt1) + (A2 × Wt2) + (A3 × Wt3) + (A4 × Wt4) + (A5 × Wt5) where A is percent area of infarction by planimetry from subscripted numbers 1-5 representing sections and Wt is the weight of the same numbered sections.Myocardial histology.
Routine histological staining was performed on multiple sections of
midventricular cardiac sections to determine the extent of PMN
infiltration. Wild-type (n = 3) and
P-selectin / (n = 4)
hearts were subjected to 30 min of myocardial ischemia and 120 min of reperfusion and stained as described above. The hearts were
stored overnight in 4.0% paraformaldehyde at 4°C. The tissue was
cut into sections and dehydrated using graded acetone washes at
4°C. Tissue sections were embedded in plastic (Immunobed;
Polysciences), and 4-µm-thick sections were cut and transferred to
Vectabond-coated slides (Vector Laboratories). The slides were soaked
in 95% ethanol for 10 min to remove some of the plastic embedding and
to allow the tissue to stain. After the 10-min ethanol wash, the tissue sections were stained with either Gill no. 3 hematoxylin solution for
10 min (Sigma) or Giemsa stain (Sigma) for 3 min. The slides were then
observed microscopically, and the number of PMNs per field of view was
counted. For each of the 3 hearts examined the number of PMNs was
counted in 4 fields for a total of 12 fields.
Myocardial P-selectin expression.
Radiolabeled P-selectin and control MAbs were prepared using the
Iodo-Gen (Sigma) method as previously described (13). Wild-type (n = 6) and P-selectin /
(n = 5) mice were anesthetized (100 mg/kg pentobarbital sodium ip) and instrumented with carotid artery and
jugular vein catheters. All animals underwent 30 min of LAD occlusion
followed by 20 min of reperfusion (Fig. 1). At 15 min of
reperfusion, radiolabeled antibodies (volume titrated to 200 µl with
0.9% NaCl) were injected. Radiolabeled
(125I) MAb directed against
P-selectin (RB40.34, PharMingen) and a nonbinding radiolabeled
(131I) antibody (P-23, Pharmacia
and Upjohn) were slowly administered through the jugular vein catheter.
After 5 min of circulation (20 min of reperfusion), a 50-µl plasma
sample was drawn. The animal was then perfused with 15 ml of warm (pH = 7.4), heparinized bicarbonate-buffered saline (BBS), while being
exsanguinated, to flush the excess P-selectin MAb and nonbinding
control antibody. LAD religation was followed by infusion of 1.0%
Evans blue dye to delineate the ischemic zone from the nonischemic
zone. The heart was then excised and serially sectioned. The ischemic
and nonischemic zones were dissected, weighed, and measured for
radioisotopic activity. Cardiac radioactivity was measured using an
automatic gamma counter (1480 Wizard, Wallac) to determine MI/R-induced P-selectin expression. Additionally, the following items were also
measured using the gamma counter: 50-µl plasma sample, syringe, intravenous catheter, and 2 µl of the original antibody mixture. The
gamma counts of these items were factored into the determination of
P-selectin expression using the following equation:
[(125I
cpm/g)/(125I cpm injected)] [(131I
cpm/g)/(131I cpm injected)].
Hematology of peripheral blood.
Total leukocyte, PMN, and platelet counts were performed by LabCorp
(BioVet Division). P-selectin /
(n = 10) and wild-type (n = 7) whole blood samples were
obtained from the carotid artery and collected into pediatric (
1 ml)
lavender-topped (EDTA containing) microtainers (Becton Dickinson).
Statistical analyses. The infarct size, AAR, LV size, leukocyte counts, and P-selectin expressions (between groups) were analyzed with a two-tailed unpaired t-test. P-selectin expression within each group (between zones) was analyzed with a paired t-test. Hemodynamic data [mean arterial blood pressure (MABP)] were analyzed with an ANOVA coupled with post hoc analysis (Scheffé test for significance). All statistics were calculated with StatView 4.5 (Abacus Concepts). All values are reported as means ± SE. Statistical significance was set at P < 0.05.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Exclusion criteria.
A total of 8 wild-type and 11 P-selectin / mice entered
the 30-min ischemia and 120-min reperfusion (30/120) MI/R
infarct size determination protocol. Two wild-type mice died early
in reperfusion, and one P-selectin / mouse died late
in reperfusion. Consequently, 6 wild-type and 10 P-selectin
/ mice successfully completed the 30/120 experimental
protocol and were included for data analysis.
/ mice
entered the 60-min ischemia and 120-min reperfusion (60/120)
MI/R infarct size determination protocol. Six wild-type mice died
during the protocol (2 in ischemia and 4 in reperfusion), and
three P-selectin / mice died during reperfusion.
Therefore, seven wild-type and seven P-selectin / mice
completed the 60/120 experimental protocol and were included for data analysis.
In the radiolabeled MAb experiments, 6 P-selectin / and
10 wild-type mice entered the protocol. One P-selectin /
and two wild-type mice were excluded because of technical problems. Two other wild-type mice did not survive the protocol because of cardiac failure. Consequently, five P-selectin / and six
wild-type mice were included for analysis of P-selectin expression
after MI/R.
Hemodynamics.
MABP was recorded for all animals in each experimental group throughout
the MI/R experimental protocol, and values are reported in Table
1 (30-min ischemia protocol) and
Table 2 (60-min ischemia protocol).
As shown in Table 1, MABP in the P-selectin / mice at 120 min of reperfusion and in the wild-type mice at baseline were
significantly (P < 0.05) lower than
in the P-selectin / at baseline. Otherwise, there were no
significant group differences in MABP observed at any time throughout
either of the experimental protocols.
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Hematology data.
The circulating levels of leukocytes, PMNs, and platelets for the
wild-type and P-selectin / mice are presented in Table 3. Total leukocyte counts were
significantly (P < 0.01) higher in
the P-selectin / mice (4,730 ± 510 cells/µl blood)
compared with the wild-type mice (2,529 ± 252 cells/µl blood).
Consequently, the number of circulating PMNs was also significantly
(P < 0.05) higher in the P-selectin
/ mice (1,630 ± 194 cells/µl blood) than in the
wild-type mice (971 ± 233 cells/µl blood). In addition, the
number of circulating platelets were significantly
(P < 0.01) higher in the P-selectin
/ mice (14,260 ± 828 cells/µl blood) than in the
wild-type mice (10,014 ± 308 cells/µl blood).
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Myocardial P-selectin expression.
A dual-radiolabeled MAb technique was used to quantify in vivo coronary
endothelial cell P-selectin expression after 30 min of coronary
ischemia and 20 min of reperfusion. P-selectin expression (µg
MAb/g tissue) was determined in nonischemic and ischemic myocardium. These data are summarized in Fig. 2.
P-selectin expression in the nonischemic zone was significantly
(P < 0.01) enhanced in the MI/R
hearts (0.037 ± 0.008 µg MAb/g tissue) compared with the
P-selectin / hearts (0.001 ± 0.001 µg
MAb/g tissue). Also, P-selectin was expressed at significantly
(P < 0.001) higher levels in the
ischemic zones of the wild-type hearts (0.070 ± 0.010 µg MAb/g
tissue) compared with the ischemic zones of the P-selectin / hearts (0.004 ± 0.004 µg MAb/g tissue). We also
measured P-selectin expression in sham-operated, wild-type mice (data
not shown) and found the constitutive expression to be significantly
lower than the nonischemic zone of the MI/R-operated wild-type mice.
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Myocardial AAR and infarct size (30-min myocardial
ischemia/120-min reperfusion).
Representative photomicrographs of AAR and infarct staining for the
30-min LAD occlusion and 120-min reperfusion protocol are presented in
Fig. 3. Despite similar-sized AAR,
wild-type hearts suffered a significantly larger area of infarction
after MI/R compared with P-selectin / hearts. Summary
data for AAR and infarct size are shown in Fig.
4. Both groups of animals experienced similar-sized ischemic zones of LV [wild-type AAR = 46.5 ± 1.1% of LV; P-selectin / AAR = 50.6 ± 3.3% of LV;
P = not significant (NS)].
Infarct size was 42.5 ± 4.4% of the AAR in wild-type mice and 24.4 ± 4.0% of the AAR in the P-selectin / mice
(P < 0.05). When the infarct size is
expressed relative to the LV, the P-selectin / mice (13.0 ± 2.4% of LV) exhibited levels of necrosis similar to those of
wild-type controls (19.9 ± 2.3% of LV,
P = NS).
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Myocardial PMN accumulation (30-min myocardial
ischemia/120-min reperfusion).
Graphic interpretation of PMN counts within the ischemic-reperfused
myocardium after 30 min of myocardial ischemia and 120 min of
reperfusion are presented in Fig. 5. The
P-selectin / hearts contained significantly fewer
(P < 0.01) PMNs than the wild-type
hearts (22.3 ± 0.9 vs. 46.5 ± 5.3 PMNs/field).
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Myocardial AAR and infarct size (60-min myocardial
ischemia/120-min reperfusion).
Representative photomicrographs for the 60/120 MI/R protocol are
presented in Fig. 6. Summary data for this
group are graphically presented in Fig. 7.
The wild-type and P-selectin / mice had similar AAR (46.0 ± 4.0% of LV and 45.1 ± 5.4% of LV, respectively; P = NS). The mean infarct size in the
P-selectin / hearts (40.4 ± 6.9% of AAR) was not
different compared with the wild-type hearts (52.1 ± 4.8% of AAR). Comparison of the area of necrosis relative to the LV
showed no difference between the P-selectin / hearts (24.5 ± 3.5% LV) and the wild-type hearts (19.3 ± 4.2% LV)
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Myocardial PMN accumulation (60-min myocardial
ischemia/120-min reperfusion).
Data for the 60-min myocardial ischemia and 120-min reperfusion
protocol are presented in Fig. 8. Again,
the P-selectin / ischemic-reperfused myocardium contained
significantly fewer (P < 0.01) PMNs
than the wild-type hearts (21.8 ± 2.4 vs. 57.3 ± 7.4 PMNs/field).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous studies laid the foundation for the mouse heart as a model for
in vivo myocardial ischemia and reperfusion injury (17, 29).
However, the present study is the first investigation of myocardial
infarct size in vivo in a P-selectin-deficient mouse. One prominent
discovery in the present study is the diminution of infarct size and
PMN accumulation in the P-selectin / mouse hearts
compared with wild-type mouse hearts. Another salient finding of this
study is the quantitative measurement of regional in vivo expression of
P-selectin after MI/R. Although other studies incorporated immunohistochemistry to qualitatively assess the level of P-selectin expression ex vivo after MI/R (36), the present study quantifies the in
vivo coronary vascular P-selectin expression in the ischemic and
nonischemic zones of the myocardium. The protective effect of
P-selectin deletion as seen in the 30-min ischemia experiments was not seen in the 60-min ischemia experiments. This is most likely caused by the fact that most of the area at risk for infarction is probably necrotic at the time of reperfusion. This does not mean
that P-selectin blockade or knockout is not protective. Instead, the
ischemic insult alone may have been severe enough to kill the tissue at
risk independent of PMN involvement.
The upregulation of P-selectin after MI/R is understood to be an early prerequisite for PMN tethering to the coronary endothelium. Previous studies (10, 11) implicated PMNs as key intermediaries of myocardial injury resulting from ischemia and reperfusion. The involvement of leukocyte-endothelial cell interactions in the coronary microcirculation after MI/R was recently demonstrated in dogs in vivo (34). PMN-mediated injury is dependent on the interaction of adhesion glycoproteins expressed on the surface of circulating PMNs (L-selectin, P-selectin glycoprotein ligand-1, and CD11/CD18) with adhesion molecules expressed on the surface of the coronary endothelium (P-selectin, E-selectin, and intercellular adhesion molecule; Refs. 12, 23). Previous investigations have shown that inhibition of P-selectin with a MAb (PB1.3) attenuated myocardial necrosis in dogs (24) and cats (36) after MI/R. Similarly, the use of a carbohydrate ligand to P-selectin (CY-1503) also diminished myocardial infarct size in dogs (15, 25, 35) and cats (5) subsequent to MI/R. Conversely, others showed that the administration of CY-1503 did not confer cardioprotection in rabbits (2) and dogs (16).
A previous study (28) using the P-selectin / mouse
demonstrated less PMN rolling and extravasation after an inflammatory stimulus. The same study also demonstrated significant neutrophilia and
thrombocytosis in P-selectin / mice under basal
conditions, both of which are confirmed in the present study. The
P-selectin / mouse is becoming a widely used tool for
demonstrating the effects of P-selectin suppression in certain
pathological states, including acute lung injury (8), carotid arterial
neointimal formation (22), cerebral stroke (7), fatty streak formation (18), and atherosclerosis (31). Much of the popularity of the
P-selectin / mouse is owed to the avoidance of problems associated with immunoneutralization such as the difficulty of investigating the role of P-selectin in chronic pathologies, the determination of antibody dose concentrations, species
cross-reactivity, time of administration, binding specificity, and
half-life. The P-selectin / mouse enables us to observe
the consequences of P-selectin deficiency in the setting of MI/R and
alleviate the aforementioned dilemmas. Therefore, the disparity between
the P-selectin / and wild-type infarct sizes and degree
of PMN infiltration is a function of acute P-selectin upregulation in
response to MI/R.
An important finding of the present study is the regional myocardial quantification of P-selectin expression. Qualitative assessment of P-selectin upregulation using immunohistochemical localization (36) indicates the relative abundance of P-selectin in the ischemic-reperfused feline myocardium. Although this powerful ex vivo technique confirms the presence of P-selectin expression, it does not quantify the level of MI/R-induced P-selectin upregulation of the in vivo coronary vasculature. P-selectin was previously quantified in vivo using the present radiolabeled MAb technique in the whole hearts of mice subjected to endotoxin or histamine stimulation (14). This previous study demonstrated profound increases in P-selectin expression throughout the myocardium 5-60 min after histamine injection and 4-8 h after endotoxin challenge. The present study differs from both of these previous studies because we have quantified the magnitude of in vivo P-selectin expression in two specific and physiologically important loci, 1) the ischemic zone and 2) the nonischemic zone. The use of this novel radiolabeled MAb technique in this manner may provide a foundation for the quantitative and spatial characterization of other ECAMs in various gene-targeted mice subjected to MI/R.
In additional experiments, we investigated the effect of prolonged
coronary artery ischemia on PMN accumulation and infarct size.
With prolonged coronary artery occlusion, we observed that infarct size
was similar in P-selectin / and wild-type mice. These
data clearly demonstrate that cardioprotection associated with
P-selectin deficiency was lost with increased duration of ischemia. This emphasizes a concept now paramount to myocardial ischemia therapy and previously demonstrated in other animals (33) and humans (3): time to reperfusion. Although infarct size
increased significantly in the P-selectin / hearts from 30-min to 60-min myocardial ischemia, there was no difference in PMN infiltration between 30-min and 60-min myocardial
ischemia (both followed by 120-min reperfusion). This suggests
that the enhanced necrosis was independent of PMNs. It is probable that the cellular death caused by the lengthened ischemia (and
independent of PMNs) was severe enough to outweigh the cardioprotection
provided by chronic P-selectin blockade, as seen in the shorter
duration of ischemia. Some of the cellular mechanisms involved
in this PMN-independent sequence of events may involve
Ca2+ overload, glycogen depletion,
serial dephosphorylation of ATP, and edema (1, 32).
Although this study has revealed significant pathophysiological
conclusions associated with P-selectin expression in the setting of
MI/R, there are several study limitations to be addressed. One
liability of our infarct size determinations is the brevity of the
reperfusion period. On the basis of previous studies it is now well
appreciated that reperfusion injury evolves during the initial
24-48 h after the initiation of reperfusion (27). Future studies
involving longer durations of reperfusion are required to further
clarify the role of P-selectin in MI/R injury. It is possible that
P-selectin and/or E-selectin may play a prominent role at later
times after reperfusion. Nonetheless, the results of the present study
provide novel insights into P-selectin and MI/R and serve as a starting
point for additional experiments in transgenic mice. Another concern is
our inability to measure coronary blood flow in mice because of
technical difficulties associated with their diminutive size. However,
it is unlikely that the observed reduction in myocardial infarct size
in P-selectin / mice is a result of greater ischemic zone
blood flow that we are at present unable to determine. Furthermore, we
did not measure postischemic myocardial contractile function in these experiments and therefore cannot determine whether the cardioprotection observed in P-selectin / mice results in improved
myocardial contractility.
In summary, using a gene-targeted knockout mouse and a novel radiolabeled MAb technique, we have clarified the in vivo coronary endothelial expression of P-selectin after MI/R and its role in the ensuing injury. We determined that PMN infiltration and infarct size were diminished in mice that did not express P-selectin. Furthermore, we have demonstrated that P-selectin deficiency may not confer cardioprotection during extended periods of coronary artery occlusion, which may be attributed to PMN-independent myocardial cell death. This is of great importance because the therapeutic efficacy of P-selectin inhibition may not be realized in subjects that experience longer periods of ischemia.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the expert technical assistance of Janice M. Russell for preparation of the radiolabeled antibodies. The authors are also thankful to DeRoyal Industries (Surgical Division, Powell, TN) and Ethicon Surgical (Somerville, NJ) for generous donations of surgical supplies.
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FOOTNOTES |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant PO1 DK-43785 (D. N. Granger) and by Grant JDF 195065 from the Juvenile Diabetes Foundation (D. J. Lefer).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: D. J. Lefer, Dept. of Molecular and Cellular Physiology, LSU Medical Center, 1501 Kings Highway, Shreveport, Louisiana 71130.
Received 7 April 1998; accepted in final form 29 July 1998.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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