© 1999 American Heart Association, Inc.
Original Contribution |
Endothelial Injuries of Coronary Arteries Distal to Thrombotic Sites
Role of Adhesive Interaction Between Endothelial P-Selectin and Leukocyte Sialyl LewisX
From the Department of Internal Medicine III (H.E., H.I., T.M., N.H., T.I.), Cardiovascular Research Institute (T.M.), Department of Pathology II (H.Y.), and Department of Internal Medicine II (S.S.), Kurume University School of Medicine, Kurume, Japan.
Correspondence to Hisao Ikeda, MD, PhD, Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. E-mail ikeikeda{at}med.kurume-u.ac.jp
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Abstract—Intracoronary thrombus formation is associated with epicardial vasoconstriction distal to the thrombotic site. To investigate the mechanisms of abnormal vasomotor function of the artery distal to the thrombotic site, we studied coronary vessels in dogs with cyclic flow variations (CFVs) of the left anterior descending coronary artery (LAD) stenosis with endothelial injury. Coronary rings isolated from the LAD (proximal, stenotic, and distal sites) and control circumflex coronary arteries were tested for responsiveness to endothelium-dependent (acetylcholine and A23187) and endothelium-independent vasodilators (NaNO2). Endothelium-independent relaxation was intact in all 4 sites. Endothelium-dependent relaxation was intact in the control and proximal sites and impaired in the stenotic sites. Relaxations not only to acetylcholine and A23187 but also to serotonin, ADP, and thrombin were impaired in the distal sites after observing CFVs for 80 minutes. Electron microscopy revealed the loss of endothelial integrity with leukocyte adherence to the endothelium in the distal sites. Immunohistochemical expression of P-selectin on the endothelial cells was more upregulated in the distal site than in the proximal site, and P-selectin mRNA expression was significantly greater in the ischemic region distal to the thrombotic site than in the proximal nonischemic region. PB1.3, a neutralizing monoclonal antibody against P-selectin, and sialyl LewisX (SLeX)–containing oligosaccharide SLeX, a carbohydrate analogue of selectin ligand, preserved endothelial function without affecting CFVs. SLeX-containing oligosaccharide preserved endothelial integrity of the distal site and inhibited P-selectin expression of the distal site. Thus, the adhesive interaction between endothelial P-selectin and leukocyte SLeX may play an important role in endothelial injuries of the coronary artery distal to the thrombotic site.
Key Words: thrombosis • P-selectin • sialyl LewisX • cell adhesion • endothelial injury
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Platelet aggregation and thrombus formation at the site of fissured atheromatous plaques are implicated as major pathogenic mechanisms underlying acute coronary syndromes.1 2 Studies in animals3 and humans4 5 have demonstrated that intracoronary thrombus formation is associated with abnormal vasomotor tone of epicardial coronary arteries distal to the thrombotic site, which may exacerbate myocardial ischemia in those syndromes. However, the precise mechanisms of abnormal vasomotor tone of coronary arteries distal to the thrombotic site remain unclear. The endothelium plays an important role in the control of vascular tone. It is then possible that the abnormal vasomotor tone distal to the thrombotic site is due to endothelial dysfunction. There is a canine model of intermittent coronary thrombus formation (the Folts coronary thrombosis model) produced by coronary artery stenosis and endothelial injury.6 We have reported that this model mimics the clinical situations of the acute coronary syndromes observed in humans.7 Accordingly, the first aim of the present study was to examine ex vivo endothelial function of the excised epicardial coronary artery distal to the thrombotic site in this canine model of the acute coronary syndromes.
Intracoronary infusion of various vasoactive substances such as serotonin causes vasoconstriction, especially in patients with coronary artery disease.8 9 It is also known that vasoactive substances are formed at the thrombotic site and released into the coronary circulation. Thus, it is considered that vasoactive substances produced at the thrombotic site contribute to the control of vasomotor tone of the distal artery and to the pathogenesis of acute coronary syndromes. Accordingly, the second aim was to examine vasoreactivity of the excised distal coronary artery to vasoactive substances such as thrombin and serotonin to further elucidate the role of thrombus formation in the pathogenesis of these syndromes.
Activated leukocytes adhere to the endothelium
and impair its function in acute ischemic
events.10 In fact, in models of
ischemia/reperfusion injury, leukocytes adhere to the
endothelium, and inhibition of the
leukocyte-endothelial interaction protects against
reperfusion-induced myocardial and endothelial
injuries.11 12 The initial process of this cellular
interaction is mediated by adhesion molecules such as P-selectin, which
is stored in both 
-granules of platelets,13 and the
Weibel-Palade bodies of endothelial
cells.14 When these cells are activated by
thrombin,15 oxygen free radicals,16 or
ischemia/reperfusion,17 P-selectin is rapidly
translocated to cell surfaces and adheres to a sialylated fucosylated
carbohydrate structure, such as sialyl LewisX
(SLeX), on leukocytes.18 19 The
functional significance of P-selectin binding to
SLeX is not completely understood. However,
several lines of evidence have indicated that activated
platelets enhance extracellular oxygen free radical generation by
leukocytes through P-selectin20 and that P-selectin
mediates "rolling" of leukocytes.21 22 In animal
models of myocardial reperfusion injury, either monoclonal antibody to
P-selectin23 or soluble
SLeX-containing oligosaccharide
(SLeX-OS)24 25 protected against
reperfusion-induced endothelial and myocardial
injuries. Thus, both in vitro and in vivo experimental studies have
illustrated that the adhesive interaction between P-selectin and
SLeX may have a critical function in modulating
vascular and tissue injuries. In patients with acute coronary
syndromes, significant increases in P-selectin expression on
platelets and a soluble form of P-selectin have been demonstrated
by our laboratory26 27 and others.28 However,
it is unknown whether this adhesive interaction between the leukocyte
and endothelium contributes to the control of
endothelial function of the coronary artery
distal to the thrombotic site in acute coronary syndromes.
Accordingly, the third aim was to examine the effects of
leukocyte-endothelial interaction on
endothelial function of coronary arteries
distal to the thrombotic site. For this purpose, we examined the
effects of PB1.3, a neutralizing monoclonal antibody against
P-selectin, and SLeX-OS, a unique carbohydrate
analogue of selectin ligand on the endothelial function
distal to the thrombotic site. Using electron microscopy, we also
examined leukocyte adherence to the coronary
endothelium and examined the expression of P-selectin
by immunohistochemical staining and with a confocal laser scanning
microscope system. We also examined P-selectin mRNA expression in
cardiac tissues of the nonischemic regions proximal to the
thrombotic site and ischemic regions distal to it.
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Materials and Methods |
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Surgical Preparation
All animal experiments were conducted in accordance with the guidelines issued from the Animal Research Committee of the Kurume University School of Medicine. Healthy mongrel dogs weighing 18 to 25 kg were anesthetized with sodium pentobarbital (30 mg/kg). They were ventilated artificially with 100% oxygen. A thoracotomy was performed in the fifth left intercostal space, and the heart was suspended in the pericardial cradle. Polyethylene catheters were placed in the right atrium for drug infusion. ECG was used to determine heart rate. A segment of the left anterior descending coronary artery (LAD) was gently dissected free from the surrounding tissue, and a pulse Doppler flow probe (Hartley Instruments) was placed proximal to the constricting cylinder. Coronary blood flow (CBF) velocity was measured with a pulsed Doppler flow system (VF-1, Crystal Biotech). Arterial blood gases and body temperature were maintained within normal physiological ranges. Aortic pressure was monitored with a micromanometer placed in the femoral artery. Heart rate, systolic and diastolic aortic blood pressure, and phasic and mean CBF velocities were continuously recorded throughout the study. An animal model of intermittent intracoronary thrombus formation, ie, cyclic flow variations (CFVs) caused by recurrent platelet aggregation and subsequent dislodgment at the coronary stenotic site with endothelial injury, were produced according to the method originally described by Folts et al.6 Briefly, after a 30-minute stabilization period, the endothelium of the exposed LAD was injured by gently squeezing the artery with cushioned forceps. Then, a cylindrical constrictor was placed around the injured coronary artery distal to the flow probe to reduce the phasic CBF velocity to
40%
of the baseline level, eliminating reactive hyperemia after 15
seconds of temporary coronary occlusion. Subsequently, CFVs
developed in 75 of 101 dogs. The remaining 26 dogs were excluded from
this study. The severity of CFVs was evaluated by monitoring mean CBF (mL/min), phasic and mean nadir CBF velocities (percentage of baseline), and the frequency (cycles per hour) for the observation period. CBF was determined according to a method described previously.7 29 Briefly, CBF velocity near the center of the vessel was recorded by using the pulsed Doppler principle, and CBF velocity was calculated by a digital planimeter. The cross-sectional area of the vessel was approximated to an inside diameter of the Doppler flow probe, ranging in size from 2.0 to 2.5 mm. Then, mean CBF was derived by multiplying mean CBF velocity by the cross-sectional area. Nadir CBF velocity was calculated by averaging the 3 lowest flow velocities and was expressed as a percentage of the unconstricted CBF velocity (baseline) according to the previous method.29 30 In dogs that exhibited only 2 flow restorations, nadir CBF velocity was calculated by averaging the 2.
Organ Chamber Experiments
After the observation period of CFVs, the heart was quickly
removed and immersed in cold, oxygenated modified
Krebs-Henseleit (K-H) solution of the following composition (in
mmol/L): NaCl 118.3, KCl 4.7,
KH2PO4 1.2,
MgSO4 1.2 CaCl2 2.5,
NaHCO3 25.0, and glucose 11.1 at pH 7.35 to 7.45.
Both the LAD and left circumflex coronary artery (LCx) segments
were carefully removed and placed into cold K-H solution. Isolated
coronary vessels were cut into rings of 2 to 3 mm in
length. The LAD rings were obtained from the stenotic site and
the proximal (10 mm apart) and distal sites (10 mm apart) of
stenosis. The LCx ring of the same dog was used as a control
vessel. Then, rings were mounted on stainless steel hooks, suspended in
2-mL tissue baths, and connected to force-displacement transducers
(UC-2, Kishimoto Medical and Chemical Industry) to record changes
in isometric force on an 8-channel recorder (Recti-Horiz-8K,
San-ei). The baths were filled with 2 mL of K-H solution and aerated at
37°C with a gas mixture of 95% O2-5%
CO2. Rings were initially stretched to give a
preload of 2.0g force and were equilibrated for 60 to 90
minutes. During this period, the K-H solution in the tissue bath was
replaced every 15 minutes. After equilibration, relaxations were
examined during a contraction caused by 10–7
mol/L U46619
(9,11-dideoxy-9,11-methanoepoxy-prostaglandin
F2), a thromboxane
A2 analogue. The LAD rings and control ring from
the same dog were examined in parallel in the 6 groups discussed below
(Figure 1
).
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Group 1
After CFVs were observed for 20 minutes, coronary rings
were isolated from dogs (n=6) that intravenously received a
bolus of saline followed by a continuous infusion of saline (1 mL per
hour). Once a stable contraction with U46619
(10–7 mol/L) was obtained, an
endothelium-dependent vasodilator, acetylcholine (ACh),
was added to the bath in cumulative concentrations at
10–9 to 10–5 mol/L. After
the responses were stabilized, rings were washed and allowed to
equilibrate to baseline once again. The procedure was repeated with
another endothelium-dependent vasodilator, the calcium
ionophore A23187 (10–9 to
10–5 mol/L), and then again with an
endothelium-independent vasodilator, acidified
NaNO2 (10–8 to
10–3 mol/L).
Group 2
After CFVs were observed for 80 minutes, coronary rings
were isolated from dogs (n=9) that received saline in the same way as
group 1. Coronary ring studies were performed in the same
manner as were those done for group 1.
Group 3
After CFVs were observed for 80 minutes, coronary rings
were isolated from dogs (n=6) that intravenously received a
bolus of PB1.3 (1 mg/kg). We previously confirmed that this dose of
PB1.3 did not abolish CFVs.29 Coronary ring
studies were performed in the same manner as were those done for group
1.
Group 4
After CFVs were observed for 80 minutes, coronary rings
were isolated from dogs (n=8) that intravenously received a
bolus of SLeX-OS (5 mg/kg) followed by a
continuous infusion at 5 mg/kg per hour. We previously confirmed that
this dose of SLeX-OS did not abolish CFVs,
although a higher dose of SLeX-OS (40 mg/kg as a
bolus injection followed by a continuous infusion at 5 mg/kg per hour)
significantly reduced CFVs.29 Coronary ring
studies were performed in the same manner as were those done for group
1.
Group 5
After CFVs were observed for 80 minutes, coronary rings
were isolated from dogs (n=7) that intravenously received a
bolus of PNB1.6, a nonblocking monoclonal antibody against P-selectin
(1 mg/kg). Coronary ring studies were performed in the same
manner as were those done for group 1.
Group 6
The methods of production of CFVs and coronary
ring isolation were similar to those used in group 2. There were 7
animals in this group. Once a stable contraction with U46619
(10–7 mol/L) was obtained, serotonin
was added into the bath in cumulative concentrations at
10–9 to 10–4 mol/L. After
the responses were stabilized, rings were washed and allowed to
equilibrate to baseline once again. The procedure was repeated with an
ADP (10–9 to 10–4 mol/L)
and then again with thrombin (0.003 to 3 U/mL).
In all experiments, indomethacin (10–5 mol/L) was added to organ baths to exclude the effect of endogenous prostanoids. Data are expressed as percentage relaxation of the contractions to U46619.
Morphological Studies
To assess morphological changes of the coronary
arteries, additional dogs were subjected to 20- or 80-minute CFVs
(n=8). After the hearts were quickly removed, a catheter was placed
into the left coronary ostium. Then, 2%
glutaraldehyde in PBS was perfused through the catheter
at 100 mm Hg pressure for 10 minutes. The LAD and LCx were
carefully dissected from the heart, and the constrictor was removed.
The segments were longitudinally dissected and visualized under a
dissecting microscope. The specimens were incubated in the same
fixation for 2 hours and rinsed with 0.1 mol/L cacodylated buffer
containing 0.1 mol/L sucrose for 12 hours. Furthermore, the specimens
were immersed in 1% osmium tetroxide for 1 hour, dehydrated in a
series of graded concentrations of cold ethanol, dried by the
critical-point drying method, mounted on silver blocks, coated with
10 nm of gold, and observed under a scanning electron microscope
(S-800, Hitachi) operated at 20 kV.
Immunohistochemical and Immunofluorescent Studies
For immunohistochemical study of the coronary arteries
using a monoclonal antibody against P-selectin, additional dogs were
subjected to 20- or 80-minute CFVs (n=9). After the hearts were quickly
removed, the LAD and LCx were carefully dissected from the heart. The
isolated coronary arteries were embedded in OCT compound and
frozen in liquid nitrogen. Serial 4-µm-thick frozen sections were
adhered to poly-L-lysine–coated slides and then fixed in
cold acetone for 10 minutes. The labeled streptavidin-biotin method was
used for immunohistochemical staining as described
previously31 (DAKO LSAB kit). Briefly, specimens were
treated with 3% hydrogen peroxide for 5 minutes to inhibit
endogenous peroxidase and then incubated with 1% BSA.
Subsequently, they were incubated with 10 µg/mL of CRC81, which is a
specific monoclonal antibody against P-selectin and does not recognize
the functional binding site of P-selectin to SLeX
(Biodesign International), or with a similar amount of nonimmune mouse
IgG for 1 hour at room temperature. After washing 3 times in PBS (pH
7.4), biotinylated anti-mouse IgG secondary antibodies were applied,
followed by peroxidase-labeled streptavidin. Peroxidase activity was
visualized with 3-amino-9-ethylcarbazole, and the sections were faintly
counterstained with Mayer's hematoxylin.
To quantify the cellular expression of P-selectin on the endothelium of the coronary arteries, additional dogs were subjected to 20- or 80-minute CFVs (n=9). In the same manner and procedure as described above, specimens were incubated with 1% BSA. Subsequently, they were incubated with 5 µg/mL of CRC81 or with a similar amount of nonimmune mouse IgG for 24 hours at 4°C. After washing 3 times in PBS (pH 7.4), they were incubated with FITC-labeled goat anti-mouse IgG antibody (Cappel Laboratories) at a 1:200 dilution with PBS at room temperature for 1 hour. They were washed with PBS and then were mounted using Vectashield (Vector Laboratories). Immunostained specimens were observed using a confocal laser scanning microscope system (LSM-GB200, Olympus) at an excitation wavelength of 488 nm and emission wavelength of 530 nm, as previously described.32 Then, the confocal images were continuously digitized into a 1024x768–pixel matrix image with 256 gray-scale levels/pixel, and the 7 rectangle regions of interest were set on the different regions of the confocal image by an independent observer (S.S.), who had no knowledge of the study protocol. In each region of interest, immunofluorescent intensity was measured by a computer-aided technique, and the averaged intensity was calculated. To correct for background intensity, the intensity at the use of a nonimmune mouse IgG was subtracted from the intensity at the use of a monoclonal antibody against P-selectin.
Northern Analysis for Canine P-Selectin mRNA Expression in
Cardiac Tissue
Digoxigenin (DIG)–labeled canine P-selectin RNA probe was
prepared by in vitro transcription from a linearized template according
to the Genius RNA probe labeling kit (Boehringer Mannheim).
Canine P-selectin cDNA cloned into pBluescript II SK+ (kindly provided
by Dr Mark L. Entman, Baylor College of Medicine, Houston, TX) was used
to generate a single-stranded antisense (3'-5') RNA probe. Before
beginning the transcription reaction, the DNA template was linearized
by digestion with a restriction enzyme, BglII, that cut
downstream of the insert to avoid transcription of undesirable plasmid
sequences. The linearized template (1 µg) was incubated in 20 mL of
1x NTP mixture (in mmol/L, ATP 1 GTP 1, CTP 1, DIG-UTP 0.35, and
UTP 0.65), T7 polymerase (2 U/mL), and DEPC-treated water for 2 hours
at 37°C.
RNA was isolated by a phenol/chloroform extraction procedure.33 Twenty micrograms of total RNA from each sample (nonischemic proximal and ischemic distal myocardium to CFVs at the thrombotic site in each of 6 dogs) were separated by electrophoresis in 1% agarose gel containing 2.2 mol/L formaldehyde. After capillary transfer to a nylon membrane (Hybond-N, Amersham), rRNA was visualized by ethidium bromide staining to verify that equal amounts of RNA had been transferred to the membrane. The membrane was prehybridized for 3 hours and hybridized overnight with DIG-labeled antisense RNA probe at 45°C. Chemiluminescence signals derived from hybridized probe were detected on an x-ray film using a DIG luminescence detection kit (Boehringer Mannheim). Obtained blots of P-selectin mRNA expression from all 6 animals were quantitatively assessed by densitometric analysis using the NIH image analysis system.
Materials
The following reagents were used: ACh, A23187,
NaNO2, ADP, serotonin, thrombin,
indomethacin (Sigma), and U46619 (Cayman Chemical). All
drugs were prepared daily with distilled water except for
indomethacin, which was dissolved in
Na2CO3
(10–5 mol/L) after sonication, and A23187, which
was dissolved in DMSO, with further dilutions obtained in distilled
water. The concentrations are expressed as the final molar
concentration in the organ bath.
Statistical Analysis
All values are presented as mean±SE; n refers to the
number of dogs from which the coronary artery was taken. The
paired t test was used for comparison of 2 means.
Repeated-measures ANOVA with the Scheffé test was applied for
multiple comparisons. Differences were considered statistically
significant when the probability was <0.05.
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Results |
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Hemodynamic Variables Before and During CFVs
Before developing CFVs (stenosis in the Table
), endothelium
injury and coronary constriction decreased the averaged peak
phasic CBF velocity to 38% to 42% of baseline and mean peak CBF
velocity to 47% to 51% of baseline. Heart rate, aortic pressure, and
peak phasic and mean CBF velocities were similar among the 6 groups.
After developing CFVs (20- and 80-minute CFVs in the Table), no
significant changes were observed in heart rates and in
systolic and diastolic aortic blood pressures. The
peak phasic and mean CBF velocities were similarly decreased among the
6 groups. The phasic nadir CBF velocity was decreased to 7% to 8% of
control and mean nadir CBF velocity to 12% to 14% of control, not
significantly different among the 6 groups. The mean CBF and frequency
was 6.5 to 6.8 mL/min and 8.3 to 8.8 cycles per hour, respectively.
These values were also similar among the 6 groups. Thus, the PB1.3,
SLeX-OS, and PNB1.6 administration did not affect
CFVs in groups 3, 4, and 5, respectively.
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Organ Chamber Experiments
In group 1 (Figure 2A), in dogs with
20-minute CFVs, both ACh and A23187 caused comparable
endothelium-dependent, concentration-dependent
relaxations in the control, proximal, and distal sites. However,
endothelium-dependent, concentration-dependent
relaxations in the stenotic sites were significantly impaired.
NaNO2 caused comparable concentration-dependent
relaxations among the 4 sites, indicating normal
endothelium-independent relaxation. In group 2 (Figure 2B), in dogs with 80-minute CFVs,
endothelium-dependent relaxations to ACh and A23187
were impaired not only in the stenotic sites but also in the
distal sites. Other vasodilator responses were similar to those in
group 1. In groups 3 and 4 (Figure 3),
treatment with PB1.3 or SLeX-OS did not affect
80-minute CFVs. Treatment with PB1.3 (Figure 3A) or
SLeX-OS (Figure 3B) reversed the impaired
endothelium-dependent relaxation of the distal
arteries. Vasodilator responses of other sites were similar to those in
group 2. In group 5 (Figure 3C), treatment with PNB1.6 did not
affect 80-minute CFVs. Treatment with PNB1.6 did not reverse the
impaired endothelium-dependent relaxation of the distal
arteries. In group 6 (data not shown),
endothelium-dependent relaxations to
serotonin, ADP, or thrombin were also significantly
impaired in the stenotic and distal sites in dogs of 80-minute
CFVs. These changes were similar to those for ACh and A23187 observed
in group 2.
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), stenotic (
), and
distal sites (
) of the LAD and from the LCx as a control vessel
(
). In dogs with 20-minute CFVs, the endothelial
function is impaired only in the stenotic sites. In dogs with
80-minute CFVs, the endothelium-dependent relaxations
are significantly impaired not only in the stenotic sites but
also in the distal sites. *P<0.05 as compared with
controls.
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Morphology of Coronary Arteries
Representative morphological data by scanning
electron photomicrography are illustrated in Figure 4. In coronary arteries
after 80-minute CFVs, the control (Figure 4A) and proximal
(Figure 4B) sites showed the intact endothelium.
The stenotic site showed mechanically damaged
endothelium with numerous platelets and leukocytes
(Figure 4C). The distal site after 20-minute CFVs (Figure 4D) showed the intact endothelium, whereas that
after 80-minute CFVs showed the loss of endothelial
integrity with adhered leukocytes on the luminal surface (Figure 4E). The endothelial integrity at the distal
site was preserved after treatment with SLeX-OS
(Figure 4F).
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Immunohistochemical Localization and Immunofluorescent
Expression of P-Selectin
Representative immunohistochemical stainings are
illustrated in Figure 5. There was only
faint patchy expression of P-selectin by endothelial
cells in the control (Figure 5A) and proximal (Figure 5B)
sites after 80-minute CFVs. In contrast, the
endothelial cell staining patterns for the distal site
after 20-minute (Figure 5C) and 80-minute (Figure 5D)
CFVs were more intense and continuous than that in the control site.
The endothelial cell staining in the distal site after
treatment with SLeX-OS (Figure 5E) was
less intense than that with saline after 80-minute CFVs (Figure 5D). No such staining was found when the specific antibody
against P-selectin was replaced by a nonimmune mouse IgG (Figure 5F). When the immunostained specimens were observed
using a confocal laser scanning microscope (Figure 6), the expression of P-selectin by the
endothelial cells in the distal site of the LAD after
80-minute CFVs (Figure 6B) was more strongly demonstrated than
that after 20-minute CFVs (Figure 6A) or after treatment with
SLeX-OS (Figure 6C). The quantitative
analysis of the extent of immunofluorescent expression
of P-selectin is shown in Figure 7. The
localization of P-selectin was observed on the
endothelium of the control, proximal, and distal sites
in the epicardial coronary arteries. The averaged intensity of
the distal site after 20-minute CFVs was significantly higher than that
of the control or proximal sites after 80-minute CFVs
(P<0.05). Furthermore, the intensity of the distal site
after 80-minute CFVs was significantly higher than that after 20-minute
CFVs (P<0.05). The intensity of the distal site after
80-minute CFVs was significantly decreased after treatment with
SLeX-OS (P<0.05).
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P<0.05 as compared with
the control and proximal sites after 80-minute CFVs, the distal site
after 20-minute CFVs, and the distal site after 80-minute CFVs treated
with SLeX-OS. Data shown are mean±SE.
Northern Analysis for P-Selectin mRNA Expression in
Cardiac Tissue
P-selectin mRNA expression was greater in the ischemic
distal region as compared with the nonischemic proximal region
in all 6 animals (Figure 8). By the
densitometric analysis, the degree of P-selectin mRNA
expression was significantly greater in the ischemic distal
region than in the nonischemic proximal region (1.56±0.25
versus 0.88±0.17 arbitrary units, P<0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is clinically well recognized that vasomotor tone of the coronary artery distal to the thrombotic site is abnormal in patients with coronary artery disease.4 5 8 9 However, the mechanisms remain unknown. The Folts coronary thrombosis model6 used in this study is characterized by CFVs.2 We have previously demonstrated that this model produces pathophysiological manifestations similar to those of acute coronary syndromes in humans, such as ST-segment elevation, depending on the fluctuation of CBF during the episode of CFVs.7 Indeed, the phenomenon of CFVs can be observed in patients with unstable angina.34 Thus, the Folts model is well established as an in vivo model of coronary arterial thrombosis.35 In the present study, we therefore focused on the mechanisms of abnormal vasomotor tone of coronary arteries distal to the thrombotic site observed in patients with acute coronary syndromes and investigated this issue by use of a well-established canine coronary thrombosis model of CFVs. In the present study, the coronary arteries distal to the thrombotic site exhibited not only impaired endothelial function but also morphological damages of endothelium with leukocyte adherence. Moreover, the immunostainings revealed the increased expression of P-selectin by endothelial cells of coronary artery distal to the thrombotic site after developing CFVs. The administration of PB1.3, a neutralizing monoclonal antibody against P-selectin, or SLeX-OS, a unique carbohydrate analogue of selectin ligand on leukocytes, which did not affect CFVs, preserved the endothelial function in the distal sites of LAD coronary artery. The SLeX-OS prevented morphological abnormalities and inhibited the expression of P-selectin. Finally, P-selectin mRNA expression was significantly greater in the ischemic region distal to the thrombotic site as compared with the proximal nonischemic region. These findings suggest that thrombosis per se or substances released from the thrombi may cause endothelial injury of arteries distal to the thrombotic site through the adhesive interaction between endothelial P-selectin and leukocyte SLeX. Our findings may give deep and new insight into the pathogenesis of unstable angina and provide in vivo evidence for vasculoprotective efficacy of PB1.3 and SLeX-OS.
Endothelial Injury Distal to Thrombotic
Site
In control dogs with 80-minute CFVs, we demonstrated that the
coronary arteries distal to the thrombotic site had
weaker vasodilator responses to ACh and A23187 than did the
proximal or control LCx arteries, whereas the vasodilator responses to
NaNO2 were similar among these 3 sites,
indicating functional impairment of the endothelium and
normal vascular smooth muscle dilator function. The impaired
endothelium-dependent relaxation of the
stenotic site was apparently due to a surgical trauma.
Endothelium-dependent relaxations of the distal
arteries to ACh and A23187 were comparably impaired. Since A23187
activates the synthesis and release of nitric oxide while
bypassing cell membrane receptors,36 the
endothelial dysfunction of the distal artery may not be
specifically receptor mediated. This functional impairment of the
endothelium of the distal artery was associated with
loss of the morphological integrity of the endothelium
with adhered leukocytes. This impairment was not due to the surgical
trauma to the endothelium, because the distal site was
at least 10 mm away from the stenotic site and because no
such damages were observed in dogs with 20-minute CFVs in which the
same surgical procedure was performed. This endothelial
damage was not caused by CFVs per se, because the
endothelium of the distal artery of 20-minute CFVs was
intact. Endothelium-dependent relaxation was impaired
in response not only to ACh but also to other vasoactive substances,
such as serotonin, ADP, and thrombin. Because these
substances are generally produced by thrombi and released into the
circulation,2 it may be assumed that, in the presence of
abnormal endothelial function of the distal artery,
vasoactive substances produced by thrombi cause more vasoconstriction
and further aggravate myocardial ischemia.
Role of Adhesive Interaction Between Endothelial
P-Selectin and Leukocyte SLeX
Leukocyte adhesion to the endothelium plays an
important role in the sequelae of myocardial
ischemia/reperfusion injury. Adhered and activated
leukocytes release a variety of cytotoxic mediators including oxygen
free radicals, inflammatory cytokines, platelet-activating
factor, leukotriene B4, and
proteolytic enzymes.37 38 39 These mediators aggravate
endothelial dysfunction, resulting in increased
leukocyte adhesion to the endothelium and myocardial
injury.10 The inhibition11 12 or
depletion40 41 of leukocytes has been demonstrated to
result in a reduction in reperfusion injury. Because
ischemia/reperfusion results in elaboration of a variety of
humoral mediators, P-selectin is expressed within minutes after
reperfusion.17 On activation of the
endothelium, P-selectin is rapidly translocated onto
the endothelial surface, where it tethers leukocytes
and activates them. In the present study, we demonstrated
the functional and morphological injuries of the
endothelium with adhered leukocytes and the upregulated
P-selectin expression on the endothelium of
coronary arteries distal to the thrombotic site after
developing CFVs. In contrast, P-selectin was only faintly expressed on
the endothelial surface of the control and proximal
sites as shown by immunohistochemistry. To further elucidate the
localization and extent of P-selectin expression in the
coronary arteries, we quantitatively assessed the
immunofluorescent expression of P-selectin by the confocal
laser scanning system (Figure 7
). The immunofluorescent
localization of P-selectin was observed on the
endothelium of the epicardial coronary
arteries. These findings are consistent with the results of
previous studies demonstrating that P-selectin is located on the
endothelium of large arteries.23 42 43 The
P-selectin expression of the distal site after 80-minute CFVs was
significantly upregulated by 5-fold as compared with that of the
control and proximal sites after 80-minute CFVs and by 2.5-fold as
compared with that after 20-minute CFVs. Thus, our findings indicate
that P-selectin exists in the endothelium of the
epicardial coronary arteries and that the expression of
P-selectin distal to the thrombotic site progressively increases
depending on the duration of CFVs.
In the present study, the functional and morphological injuries to the endothelium distal to the thrombotic site after 20-minute CFVs were not observed despite the increased expression of P-selectin at this time point. There may be several possible reasons. First, there may exist a time difference between the expression of P-selectin and endothelial dysfunction. Firm attachment of leukocytes to the endothelium is necessary to induce endothelial injuries.10 12 P-selectin supports leukocyte tethering and rolling at the early phase of leukocyte-endothelial interaction,21 22 and then endothelial intercellular adhesion molecule-1 interacts with ß2 integrin on the leukocytes to further strengthen the adhesive interaction.44 Thus, even when P-selectin is significantly upregulated at 20 minutes after developing CFVs, endothelial dysfunction may not be necessarily induced at this time point. Another possibility is that endothelial dysfunction may be quantitatively related to the extent of P-selectin expression on the endothelial cells. More enhanced P-selectin expression could recruit more leukocytes to the coronary endothelial cells distal to the thrombotic site. In the present study, the extent of P-selectin expression was 2.5-fold more at 80 minutes than at 20 minutes after developing CFVs. Therefore, the severity of endothelial dysfunction may be related to the magnitude of P-selectin expression.
In the present study, several possible mechanisms of the expression of P-selectin distal to the thrombotic site are considered. Our total experimental period is 80 minutes. Hence, it is more likely that upregulated P-selectin on coronary endothelium at the distal site is related to externalization of P-selectin from the Weibel-Palade bodies, because de novo protein synthesis generally requires 3 to 6 hours.45 46 We examined whether P-selectin mRNA in the cardiac tissues distal to the thrombotic site might be stimulated within the observation period of 80-minute CFVs. Consequently, the degree of P-selectin mRNA expression was significantly greater in the ischemic distal region by 1.8-fold as compared with that of the nonischemic proximal region. Thus, these findings suggest that de novo transcription and protein synthesis of P-selectin may modulate the disease process of thrombus formation in the later phase of CFVs, although this possibility is less likely in our 80-minute CFVs model.
If CFVs were abolished during PB1.3 or SLeX-OS administration, the improvement of coronary endothelial dysfunction could be attributed to the disappearance of CFVs by the treatment per se rather than by the direct effect of these agents on the endothelial dysfunction. To exclude this possibility, we chose a dose of 1 mg/kg as a bolus injection of PB1.3 and a dose of 5 mg/kg as a bolus injection followed by a continuous infusion at 5 mg/kg per hour of SLeX-OS, because we have previously confirmed that these treatments did not reduce CFVs.29 In this study, it should be noted that the administration of PB1.3 and SLeX-OS significantly restored endothelial function without affecting CFVs, whereas the administration of PNB1.6 did not. Furthermore, the administration of SLeX-OS inhibited the expression of P-selectin and prevented morphological damages of the endothelium. These findings suggest that P-selectin of the endothelium of the artery distal to the thrombotic site is upregulated and traffics leukocytes that further damage the endothelium. Although our animal model is intermittent ischemia/reperfusion, the endothelial damages were not attributed to ischemia/reperfusion per se, because both PB1.3 and SLeX-OS protected the endothelium without affecting CFVs. Thus, the endothelial damage in this model was caused by mechanisms related to thrombosis rather than by ischemia/reperfusion itself. In this regard, our findings are different and new as compared with previous studies using ischemia/reperfusion models.
We think that thrombus formation, or certain substances released from thrombi, might have caused the expression of P-selectin and endothelial damages. Numerous leukocytes with platelet thrombi have been shown to be present at the stenotic site in dogs with CFVs.47 It has been shown that activated platelets induce the production of oxygen free radicals by leukocytes through platelet P-selection,20 and oxygen free radicals have an active role in initiating or sustaining CFVs.48 49 50 As discussed above, oxidative stress upregulates the expression of endothelial P-selectin.16 Thus, it is possible that platelet P-selectin–mediated, neutrophil-induced oxygen free radicals might have induced endothelial expression of P-selectin and that effects of PB1.3 and SLeX-OS may have been mediated by interruption of the platelet-leukocyte interaction. However, the contribution of platelets is unknown from our study, although platelets were present with leukocytes on the damaged endothelium of the stenotic site. Another candidate is thrombin, which causes platelet aggregation and induces the expression of P-selectin by the endothelial cells.15 Because thrombin is an important mediator of CFVs51 and its levels appear to be quite high in the present model, thrombin might have induced endothelial expression of P-selectin.
The underlying mechanisms by which SLeX-OS
decreases P-selectin expression and improves
endothelial dysfunction are still unknown. The
following explanation is considered. The protective effect of
SLeX-OS against endothelial
dysfunction may be explained by the inhibition of P-selectin–mediated
leukocyte adherence to the endothelium by attenuating
native SLeX binding to P-selectin.24
Because adhered and activated leukocytes release a number of
cytotoxic substances, such as oxygen free radicals and inflammatory
cytokines, these substances stimulate
endothelial P-selectin expression.16 52 In
the present study, SLeX-OS treatment clearly
inhibited leukocyte adherence to the endothelium distal
to the thrombotic site in the canine coronary artery, as
presented by our electron micrography (Figure 4), which
could lead to the decrease in P-selectin expression of the
endothelium.
Limitations
There are 2 limitations to this study. First, because
SLeX-OS blocks the interaction not only between
P-selectin and SLeX but also between other
selectin families present on the endothelium or
platelets and SLeX on leukocytes, its
vasculoprotective effect may not have been specific for P-selectin.
However, the neutralizing action of PB1.3 is specific for P-selectin,
because it does not cross-react with other selectin
families.53 Furthermore, PB1.3 used in this study is of
IgG1 isotype and reacts with P-selectin on the activated
platelet surface of not only humans but also other
mammalians.23 53 Thus, vasculoprotective effects in this
study are likely due to the inhibition of interaction between
endothelial P-selectin and leukocyte
SLeX. Second, although
SLeX-OS does not perform leukopenic
actions,24 this mechanism was not excluded in this study
because we did not count the number of leukocytes.
Clinical Implications
Several previous studies have demonstrated in dogs with CFVs that
vasoactive substances including serotonin,54
ADP,55 and thrombin51 are important mediators
of CFVs and that serotonin concentration increases by
18-fold at the stenotic site during the episode of
CFVs.54 These substances, which induce platelet
aggregation, exert vasoconstriction when the
endothelium is injured. In this study, the
coronary arteries distal to the thrombotic site showed impaired
endothelium-dependent relaxation to
serotonin, ADP, and thrombin. These findings may explain
the results of previous studies demonstrating that the coronary
arterial diameter distal to the stenosis decreases
during CFVs and increases after treatments with a
serotonin-receptor antagonist.3 We
have recently shown that a high dose of SLeX-OS
abolished CFVs in a canine model of coronary
thrombosis.29 Furthermore, a small sugar moiety such as
SLeX has low antigenicity when used in vivo and
has a potential efficacy after oral administration.56
Thus, SLeX-OS may be an important therapeutic
candidate for unstable angina from the point of view of not only
antithrombotic but also vasculoprotective effects.
Conclusions
We have demonstrated that the time-dependent impairment of
endothelium-dependent relaxation occurs in the
coronary arteries distal to the thrombotic site and that this
impairment is possibly caused by the adhesive interaction between
endothelial P-selectin and leukocyte
SLeX. To our knowledge, this is the first in vivo
study investigating the mechanisms of coronary
thrombosis-mediated endothelial dysfunction. An
analogue of SLeX, SLeX-OS,
may be useful for prevention of vascular injuries distal to the
thrombotic site in patients with acute coronary syndromes. This
issue should be further studied in humans.
|
Acknowledgments |
|---|
This study was supported in part by a grant-in-aid for scientific research (08670836) from the Ministry of Education, Science, Sports and Culture of Japan; by a research grant from the Foundation for the Advancement of Clinical Medicine (Fukuoka, Japan), and by a research grant from the Kimura Memorial Heart Foundation, Kurume, Japan. We are grateful to Dr Mark L. Entman (Baylor College of Medicine, Houston, TX) for the generous gift of the plasmid containing canine P-selectin cDNA. We gratefully acknowledge the generous supply of PB1.3, PNB1.6, and SLeX-OS from Dr Shinichiro Tojo of Sumitomo Pharmaceutical (Osaka, Japan). We also thank Drs Minoru Morimatsu and Kyuichi Tanikawa of Kurume University School of Medicine for the use of their electron microscopy system and confocal laser scanning microscope system. We are also very grateful to Kimiko Kimura and Aya Shimizu for their technical assistance.
Received October 27, 1998; accepted December 16, 1998.
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References |
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