© 1997 American Heart Association, Inc.
Articles |
The Antiadhesive and Antithrombotic Effects of the Nitric Oxide Donor SIN-1 Are Combined With a Decreased Vasoconstriction in a Porcine Model of Balloon Angioplasty
From the Laboratory of Experimental Pathology (P.P., Y.M.), Montreal Heart Institute, the Laboratory of Cell Biology of Hypertension (J.T.), Research Center, Hôtel-Dieu de Montréal, and the University of Montreal, Montreal, Quebec, Canada.
Correspondence to Yahye Merhi, Montreal Heart Institute, 5000 Belanger Street East, Montreal, Quebec H1T 1C8, Canada.
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Abstract Nitric oxide has been reported to modulate platelet and neutrophil interactions with the arterial wall. In this study, we investigated the effects of the nitric oxide donor 3-morpholino-sydnonimine (SIN-1) on platelet and neutrophil adhesion, and the vasomotor response, in a porcine model of angioplasty. Carotid arterial injury was produced by balloon dilation in control (n=10) and treated (SIN-1; 10 µg/kg + 1 µg/kg/min, IV) (n=8) pigs. At the site of deep arterial injury, the average platelet adhesion was 53.6±11.3x106/cm2 in the control animals and was significantly inhibited by more than 70%, to 15.1±4.1x106/cm2 (P<.01), by SIN-1. Neutrophil adhesion was also decreased by SIN-1, from 255.9±29.7 to 101.8±19.7x103/cm2 (P<.001). Mural thrombosis was found in 12 (71%) of the 17 injured arteries in the control group but in only 2 (17%) of the 12 injured arteries in the SIN-1–treated group (P<.05). Concomitantly, SIN-1 reduced platelet and neutrophil adhesion to the site of endothelial injury distally. The internal diameter of the carotid arteries was similar between the two groups before dilation but was 40% greater at the site of endothelial injury distally in SIN-1–treated animals after dilation (P<.05), as compared with controls. Accordingly, postangioplasty vasoconstriction was significantly attenuated from 46.3±2.9% in control pigs to 32.5±4.8% (P<.05) in SIN-1–treated animals. The beneficial effects of SIN-1 were associated with inhibition of neutrophil-mediated whole blood aggregation and of neutrophil-endothelium interactions. The potent antiadhesive and antithrombotic properties of SIN-1 in vivo were confirmed in ex vivo superfusion experiments. These results indicate that administration of a nitric oxide donor may be effective in preventing the acute pathophysiological responses to arterial injury by angioplasty.
Key Words: carotid arteries • thrombosis • nitric oxide • blood platelets • neutrophils
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Introduction |
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Angioplasty is a widely used procedure for the treatment of occlusive vessel disease. However, the occurrence of complications such as mural thrombus formation, vasoconstriction, and restenosis may limit the benefit of the procedure.1 Balloon dilation most often leads to deep arterial wall injury characterized by rupture of the internal elastic lamina and exposure of highly thrombogenic subendothelial structures, resulting in local platelet adhesion and thrombus formation.2,3 A localized vasoconstrictive response is also observed at the site of endothelial denudation induced by the distal tapering end of the balloon and has been related directly to the extent of platelet adhesion.4 Local platelet activation and release of bioactive substances may further enhance platelet aggregation and vasoconstriction.5
In addition to platelets, neutrophils have been reported to be activated after angioplasty6 and to interact with the balloon-injured artery in vivo.3,7-9 In a porcine model of balloon angioplasty, neutrophil depletion was associated with a significant decrease in postangioplasty vasoconstriction.7 We also recently reported the efficacy of a dual lipoxygenase/cyclooxygenase inhibitor8 and a selective leukotriene biosynthesis inhibitor (unpublished data, Provost et al. 1996) to prevent mural platelet and neutrophil adhesion and vasoconstriction after arterial injury by angioplasty. These beneficial effects were predominantly related to inhibition of neutrophil function, suggesting that neutrophils may play an important role in mediating thrombus formation and the vasomotor response of the balloon-injured artery in vivo.
Nitric oxide has been shown to inhibit platelet adhesion10 and aggregation.11 More recently, we demonstrated that nitric oxide, which inhibits neutrophil aggregation,12 also modulates neutrophil adhesion to the exposed arterial media13 and to the vascular endothelium14 under arterial blood flow conditions. In addition to maintaining a constant vasodilator tone, nitric oxide thus appears to confer important antiadhesive properties to the normal functioning endothelium. However, the local loss of endothelium and cessation of local nitric oxide production after arterial injury may enhance platelet and neutrophil-vessel wall interactions and promote vasoconstriction. Therefore, we investigated the effects of the nitric oxide donor SIN-1 on these pathophysiological responses after arterial injury by balloon angioplasty.
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Methods |
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Animal Preparation
Eighteen normal cross-breed Yorkshire pigs of either sex (mean weight, 17.4±0.8 kg) were prepared in accordance with the guidelines of the Canadian Council on Animal Care regulations. The animals were sedated by intramuscular injection of 225 mg ketamine (Rogarsetic, Rogar/STB Inc) and 125 mg azaperone (Stresnil, Janssen Pharmaceutica), intubated, and mechanically ventilated with ambient air. Anesthesia was maintained with 0.5% halothane (Fluothane, Ayerst).
Isolation and Labeling of Platelets and Neutrophils
Twenty to 24 hours before the experiment, 90 mL of autologous
blood anticoagulated with acid-citrate-dextrose (ACD) was used to
obtain a platelet-rich plasma by differential
centrifugation, as described
previously.3,7,8,14,15 This platelet suspension was
incubated with 382±6 µCi (14.2±0.2 MBq) 51Cr (Merck
Frosst Canada Inc) for 40 minutes. The suspension was then
centrifuged to remove unbound 51Cr, and the
radiolabeled platelets were resuspended in plasma and reinjected
into the animal. The average labeling efficiency of 51Cr
platelets was 62.8±3.7% and 57.1±4.7%, the amount of
51Cr activity injected was 241±16 µCi (8.9±0.6 MBq) and
217±17 µCi (8.0±0.6 MBq), the average specific activity of
51Cr in blood was 1.21±0.35 and
1.52±0.18x105 platelets per count per minute, and
free circulating 51Cr was only 3.7±0.9% and 5.1±0.7% in
the control and SIN-1–treated groups, respectively.
On the day of the experiment, 50 mL of autologous blood anticoagulated with ACD was collected for isolation and radiolabeling of neutrophils, as detailed previously.3,8,9,14,15 The neutrophil suspension was incubated with 424±9 µCi (15.7±0.3 MBq) 111In-tropolone (Merck Frosst Canada Inc) for 30 minutes. The suspension was then centrifuged to remove unbound 111In, and the radiolabeled neutrophils were resuspended in plasma and reinjected into the animal. This procedure yielded a neutrophil preparation that was over 95% pure and 90% viable, as assessed by the trypan blue exclusion test. The average labeling efficiency of 111In neutrophils was 85.2±4.6% and 92.9±3.5%, the amount of 111In activity injected was 354±23 µCi (13.1±0.8 MBq) and 406±20 µCi (15.0±0.7 MBq), the average specific activity of 111In in blood was 163±22 and 138±26 neutrophils per count per minute, and free circulating 111In was only 6.9±1.8% and 4.4±0.9% in the control and SIN-1–treated groups, respectively.
Carotid Arterial Injury
One hour before the angioplasty procedure, animals received
either SIN-1 (10 µg/kg bolus followed by a continuous infusion
of 1 µg/kg/min, IV, n=8) or saline vehicle control
(n=10). In some experiments, this dose of SIN-1 was found to prolong
ear bleeding time by 1.4-fold. Carotid arterial injury was
performed by using a 7F polyethylene balloon dilation catheter (size,
8 mm x 3 cm, Meditech Inc), as described
previously.3,7-9 After a single bolus of heparin (100
IU/kg, IV), the catheter was advanced under fluoroscopic control
through the right femoral artery into the left and the right common
carotid arteries between the fifth and fourth cervical vertebra. Five
inflations were performed at 6 atm pressure, each for 30 seconds with
60-second intervals between inflations. The
vasoconstrictive response localized at the site of the
distal tapering end of the balloon, where selective
endothelial injury occurred without any balloon
stretching of the arterial wall, was quantified
angiographically as detailed previously.3,7-9 In all pigs,
angiograms of the common carotid arteries were obtained before and 1
minute after angioplasty with selective intra-arterial
injection of 6.0 mL iothalamate meglumine 30% (Conray 30, Mallinckrodt
Medical Inc) diluted with 6.0 mL of physiological
saline. To calculate the degree of vasoconstriction, the lumen diameter
was measured at baseline and after balloon dilation at the site of the
greatest narrowing at the distal tapering end of the balloon. The
vasoconstriction was defined as the lumen diameter (measured with an
electronic caliper to the nearest 0.1 mm) on the postdilation
angiogram, expressed as percentage of the lumen diameter on the
predilation angiogram.
Quantification of Platelet and Neutrophil Adhesion
Immediately after the angioplasty procedure, the animals were
prepared for perfusion-fixation of the carotid arteries in situ with a
buffered solution of 2% glutaraldehyde and 1%
paraformaldehyde, perfused in an anterograde
fashion approximately 1 hour after the last angiogram. The fixed
carotid arteries were then removed and cleaned of all adventitial
tissue. The dilated portion was divided into two to three segments, and
the internal diameter and length of each segment were measured with an
electronic caliper to determine the surface area (in square
centimeters). Segments were also taken from the distal vasoconstricted
region, where selective endothelial injury was induced
by the tapering end of the balloon, and from the distal uninjured
artery, as reported previously.4 After surface
measurements, the radioactivity of each segment, as well as that of
reference blood samples, was counted for 5 minutes in a gamma counter
(Minaxi 5000, Packard Instruments Co) equipped with a computer and a
multinuclide analysis program. Knowing blood platelet and
neutrophil counts, and the radioactivity of each radionuclide in blood
and on the arterial segments, platelet
(x106) and neutrophil (x103) adhesion per
square centimeter on each segment was calculated, as detailed
previously.3,7-9
Histologic Analysis
After radioactivity counting, representative
serial 2- to 3-mm sections from each dilated arterial
segment (4 to 8 in each artery) were processed and embedded in
paraffin. Cross-sections were mounted and stained with Movat
pentachrome stain, which produced intense staining of the
internal and external elastic lamina. All specimens were evaluated for
the presence of deep arterial wall injury, which is
characterized by the presence of tears through the internal elastic
lamina with the exposure of the arterial
media.2 Deep arterial injury was found in 17 of
20 dilated arteries in control pigs and in 12 of 16 dilated arteries in
SIN-1–treated animals.
Morphometric analysis was performed on each section of the deeply injured arteries to quantify the extent of injury. The number of internal elastic laminal tears and the arc length of the internal elastic laminal fracture (fracture length, FL), traced from one dissected laminal end to the other, were used as a measure of the extent of injury. The circumferences demarcated by the internal and external elastic lamina were also measured and the ratios of FL-to-external elastic lamina (FL/EEL) and of FL-to-internal elastic lamina (FL/IEL) were calculated to correct for vessel size.
The injured arterial segments were also examined by light microscopy at low-power magnification for the presence of mural thrombus formation.
Superfusion Experiments
The direct antiadhesive effects of SIN-1 on platelets and
neutrophils were assessed ex vivo using Plexiglas superfusion flow
chambers, which mimic the tubelike or cylindrical shape of blood
vessels. In this chamber, a circumferential portion of the tubular flow
channel is replaced by the test vessel wall surface, which becomes
directly exposed to the flowing blood. These perfusion chambers have
been used previously to study platelet as well as neutrophil
interactions with the arterial media or
endothelium under flow
conditions.9,14,15
In this bioassay setup, the superfusion flow chambers were placed in parallel, in a thermostatically controlled water bath at 37°C, to allow simultaneous parallel pairwise superfusion of porcine arterial segments to porcine arterial blood before and 1 hour after intravenous administration of SIN-1. In these experiments, arterial blood from the femoral artery was circulated through superfusion chambers (2.0 mm ID, 25 mm in length), exposing arterial media, simulating deep arterial wall injury, or normal segments with intact endothelium for 5 minutes at a constant flow of 20 mL per minute (regulated by a peristaltic pump) at 37°C and then recirculated back into the animal through the femoral vein.
At the end of the superfusion experiments, the amount of platelets and neutrophils that interacted with the exposed arterial surface was quantified by counting the specific 51Cr or 111In radioactivity associated with the arterial surface, as described above.
Aggregation Studies
Whole blood aggregation was performed with an impedance
aggregometer (Type 500, Chronolog Corp)16 and fresh
arterial blood samples obtained before and 1 hour after the
administration of SIN-1. Aggregation was induced by adding to 450 µL
of blood, either 50 µL of the neutrophil agonist
N-formyl-methionyl-leucyl-phenylalanine (FMLP;
2x10-7 mol/L) (Sigma Chemical Co), or 50 µL of
the platelet agonist ADP (1x10-5 mol/L) (Sigma
Chemical Co). All studies were performed within the first minute after
blood sampling. Whole blood aggregation was automatically quantified
(amplitude, in ohms) with Aggro/link software (Chronolog
Corp).7,8,15
Determination of Platelet cGMP
The effect of SIN-1 on platelet cGMP levels was
determined in suspensions of porcine platelets
(5x108/mL) incubated with SIN-1 (1x10-6
mol/L, 1x10-5 mol/L, or
1x10-4 mol/L) or saline vehicle in the presence of
3-isobutyl-1-methylxanthine (IBMX, 1x10-4 mol/L
final concentration) (Sigma Chemical Co) for 1 hour at 37°C. Ice-cold
EDTA (1x10-3 mol/L final concentration) (Sigma
Chemical Co) was then added, and the platelet suspensions were
centrifuged at 7000g for 5 minutes at 4°C. The
supernatants were kept at -70°C until analyzed. After
acetylation of the samples, cGMP levels were determined by
radioimmunoassay.56
Statistical Analysis
Results are expressed as mean±SEM. Intergroup analyses
were performed by Student's unpaired t test, and intragroup
comparisons were assessed by Student's paired t test. The
incidence of mural thrombosis was compared by the 
2
test. Differences were considered statistically significant at
P<.05.
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Results |
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Hematologic parameters of control and SIN-1–treated pigs are shown in Table 1
. Hematocrit and
hemoglobin concentrations and blood erythrocyte, platelet,
leukocyte, and neutrophil counts were similar between the two groups.
Administration of SIN-1 led to a 19% reduction in mean
arterial blood pressure (P<.05), with a slight
increase in heart rate, from 135±11 to 151±10 beats per minute
(P<.05).
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Histologic Analysis
Results from the quantitative morphometric analysis of the
deeply injured carotid arteries by angioplasty are shown in Table 2. Rupture of the internal elastic lamina
with exposure of the arterial media was found in 92.9% and
93.0% of the arterial segments analyzed in the
control and SIN-1–treated groups, respectively. The number of internal
elastic laminal tears and the FL of the internal elastic lamina were
also similar between the two groups. In addition, the FL/EEL and FL/IEL
ratios were not different. These data indicate that the extent of deep
arterial injury was comparable between the control and
SIN-1–treated groups.
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Platelet and Neutrophil Adhesion
After balloon angioplasty, rupture of the internal elastic lamina
with exposure of the arterial media was characterized by
extensive platelet and neutrophil adhesion as shown in Figs 1 and 2.
The average platelet adhesion at the site of deep
arterial injury was
53.6±11.3x106/cm2 in the control group (Fig 1). Administration of the nitric oxide donor SIN-1 significantly
reduced this mural platelet adhesion by 71%, to
15.1±4.1x106/cm2 (P<.01).
Neutrophil adhesion at the site of deep arterial wall
injury was also significantly decreased by more than 60% by treatment
with SIN-1, from 255.9±29.7x103/cm2 in the
control group to 101.8±19.7x103/cm2
(P<.001) (Fig 2).
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In the control group, mural thrombosis at the site of deep arterial injury was characterized by a heavy thrombotic matrix, mainly composed of platelets and leukocytes, and was found in 12 (71%) of the 17 injured carotid arteries. However, in the SIN-1–treated group, mural thrombosis developed in only 2 (17%) of the 12 injured arteries (P<.05 versus control) and was characterized by a smaller thrombotic matrix.
In addition to its potent antithrombotic properties, SIN-1 inhibited
platelet adhesion to the site of endothelial
denudation induced by the distal tapering end of the balloon by more
than 92%, from 5.5±1.9 to 0.4±0.2x106/cm2
(P<.05) (Fig 1
). Platelet adhesion to distal uninjured
carotid arterial segments with intact
endothelium was low and was not significantly
influenced by SIN-1 (from 0.38±0.19 to
0.11±0.08x106/cm2; P=NS). This is
similar to our previously reported value of <0.5x106
platelets/cm2 obtained in vivo on the intact
endothelium.3,7 The nitric oxide donor also
reduced neutrophil adhesion to endothelially injured
segments (from 53.9±12.8 to
24.1±8.3x103/cm2; P=.09) and to
distal uninjured arterial segments with intact
endothelium (from 23.5±4.7 to
7.8±1.8x103/cm2; P<.01).
Postangioplasty Vasoconstriction
After balloon dilation, a localized
vasoconstrictive response was observed at the distal
edge of the dilated area, where selective endothelial
injury without arterial wall stretching was induced by the
tapering end of the balloon.3,7-9 This vasoconstriction was
significantly attenuated by 30% in SIN-1–treated animals (Table 3). The lumen diameter of the carotid
arteries, which was similar between the two groups before dilation, was
40% greater in SIN-1–treated animals after dilation, as compared with
controls.
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Ex Vivo Superfusion Experiments
In ex vivo experiments, the antiadhesive properties of SIN-1 were
examined using superfusion flow chambers, which mimic the cylindrical
shape of blood vessels, and fresh nonanticoagulated porcine
arterial blood. In these experiments, platelet adhesion
on the exposed arterial media was reduced by 63%, from
42.3±15.0 to 15.6±3.1x106/cm2, following
intravenous administration of SIN-1. Neutrophil adhesion on
the exposed media was almost 70% lower, 8.8±0.1 versus
28.6±4.8x103/cm2, after SIN-1 treatment. The
average neutrophil adhesion to arterial segments with
intact endothelium was
3.3±1.1x103/cm2 and was completely inhibited
by infusion of the nitric oxide donor. SIN-1 did not alter the
endothelial resistance to platelet adhesion.
Whole Blood Aggregation
Whole blood aggregometry was performed to examine the effects of
SIN-1 on whole blood aggregation induced by specific platelet and
neutrophil agonists. Whole blood aggregation induced by the neutrophil
agonist FMLP was inhibited by more than 60%, from 11.1±1.4 to
4.1±2.3 ohms (P<.05), by the nitric oxide donor SIN-1,
whereas that elicited by the platelet agonist ADP was slightly, but
not significantly, attenuated, from 19.6±1.1 to 16.8±2.1 ohms
(P=.08).
Platelet cGMP
In vitro, the nitric oxide donor SIN-1 exerted a dose-dependent
stimulatory effect on platelet cGMP production, as shown in
Table 4. Increases of 25-, 46-, and
69-fold in platelet cGMP was observed after incubation with
increasing concentrations of SIN-1.
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Discussion |
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Deep arterial wall injury related to balloon angioplasty is characterized by rupture of the internal elastic lamina with exposure of the highly thrombogenic arterial media at the site of dilation,1,2 which favors platelet and neutrophil adhesion and mural thrombus formation.2,3 Administration of the nitric oxide donor SIN-1 led to a significant reduction in platelet and neutrophil-vessel wall interactions with the balloon-injured artery, which was associated with a significant decrease in vasoconstriction at the site of endothelial injury distally.
Nitroglycerin has been reported to reduce platelet adhesion and vasoconstriction after experimental angioplasty.17 However, this organic nitrate requires a complex enzymatic biotransformation involving thiol groups to exert full biological activity.18 In addition, the efficacy of organic nitrates has been shown to be abrogated by the development of tolerance. SIN-1 is the active metabolite of the nitrovasodilator molsidomine19 and releases nitric oxide by spontaneous hydrolysis,20 rather than by enzyme action, which may explain the lack of tolerance to this agent in experimental models.21 In vivo, both SIN-122 and molsidomine19 have been found to exert potent antiplatelet effects, as demonstrated by their ability to inhibit cyclic flow variations in stenosed canine coronary arteries.
In this study, the nitric oxide donor SIN-1 significantly reduced platelet adhesion and mural thrombosis at the site of deep arterial wall injury in vivo. Similar antiadhesive and antithrombotic effects of SIN-1 were observed in ex vivo superfusion experiments performed under well-controlled flow conditions. The possible influence of any hemodynamic changes induced by SIN-1 on its antiadhesive and antithrombotic potency in vivo thus appears to be unlikely. At the dose used in the present study, the plasma concentration of SIN-1 is estimated in the low micromolar range. The antiadhesive effect of SIN-1 at this concentration was associated with a significant increase in platelet cGMP generation in vitro, which is known to mediate the inhibitory effect of nitric oxide on platelets.57 Groves et al23 also reported the antithrombotic efficacy of SIN-1 in vivo in parallel with an increase in platelet cGMP levels but not with an inhibition of ex vivo platelet aggregation in whole blood. Similarly, we found no significant changes in whole blood aggregation in response to the specific platelet agonist ADP after SIN-1 administration. Although similar findings have been obtained by other groups using the nitric oxide donors SIN-124 or pirsidomine,25 we cannot eliminate the possibility of an antiaggregatory effect of SIN-1 in vivo, because the contribution of the vessel wall substrate exposed and of local shear rate is not examined in aggregation studies in vitro. In this study, endothelial denudation without exposure of the arterial media was associated with a monolayer of adherent platelets, as reported previously.3 In vivo, SIN-1 almost completely abolished platelet adhesion to the site of endothelial denudation, where platelet-vessel wall interactions are predominant. Because platelet adhesion to the injured arterial wall initiates the thrombotic process, it may be that inhibition of platelet adhesion could limit the recruitment of other platelets, thereby preventing mural thrombosis. An additional mechanism by which SIN-1 may have reduced mural thrombus formation is through its potential ability to inhibit the release of plasminogen activator inhibitor type I from activated platelets,26 thereby enhancing endogenous fibrinolysis.
Nitric oxide has been shown to modulate neutrophil adhesion to intact14 and deeply injured arteries13 and to inhibit aggregation of isolated neutrophils in vitro.12 Because neutrophils may play an important role in the pathophysiological response to arterial injury in vivo,3,7,8 the neutrophil-inhibitory effects of the nitric oxide donor SIN-1 were examined. In the study detailed herein, SIN-1 significantly reduced neutrophil adhesion on deeply injured arterial segments by angioplasty, which was confirmed in the ex vivo superfusion experiments. These observations support our previous finding that nitric oxide modulates neutrophil interaction with the arterial media under flow conditions.12 In addition, whole blood aggregation induced by the specific neutrophil agonist FMLP, which lacks direct platelet-stimulating effect,27 was reduced by more than 60% by SIN-1. Recently, it has been shown that neutrophil depletion by treatment with cyclophosphamide almost completely inhibited whole blood aggregation induced by FMLP, without affecting blood platelet count, function, and aggregation in response to ADP.7 Also, neutrophils stimulated by FMLP have been reported to induce aggregation and thromboxane formation in co-incubated platelets in vitro.28 Together, these findings suggest that the efficacy of the nitric oxide donor SIN-1 in vivo may be related to its ability to prevent neutrophil activation and neutrophil-mediated aggregation.
Recent studies have described the inhibitory effects of nitric oxide donors on neutrophil function in vitro. The nitric oxide donors GEA 3162 and GEA 5024 have been shown to inhibit A23187-stimulated leukotriene (LT) B4 and ß-glucuronidase release, FMLP-induced chemotaxis, and opsonized zymosan-triggered superoxide anion production in human neutrophils.29 Riutta et al30 demonstrated that LTB4 production was inhibited by SIN-1 but was not affected by the cGMP analogue dibutyryl cGMP in human polymorphonuclear leukocytes. These observations are consistent with previous studies reporting inhibition of lysosomal enzyme release31 and LTB4 production32 by the nitric oxide donor SIN-1 in FMLP-stimulated neutrophils. In our study, the neutrophil inhibitory effects of SIN-1 could not be correlated with changes in cGMP levels, because porcine neutrophils did not release detectable amounts of cGMP (unpublished data, Provost et al. 1996). However, although the ability of nitric oxide donors to inhibit lysosomal enzyme release seems to be mediated by cGMP,29,31 inhibition of neutrophil LTB4 production seems to be independent of cGMP and is probably caused by nitric oxide.30,32 This effect of nitric oxide on arachidonic acid metabolism may be important in view of the implication of neutrophils in the pathophysiological response to arterial injury3,7-9 and the efficacy of leukotriene inhibition in our porcine model of balloon angioplasty.8
Recently, an increase in neutrophil adhesion to distal uninjured arterial segments with intact thromboresistant endothelium has been reported after deep arterial injury by angioplasty in pigs.3 In the present study, neutrophil adhesion to distal arterial segments with intact endothelium was reduced by nearly 70% by SIN-1. We also found that neutrophil adhesion to the endothelium was completely inhibited by SIN-1 in ex vivo superfusion experiments. These results are in accordance with our previous findings that endothelium-derived nitric oxide modulates neutrophil adhesion to the endothelium under arterial flow conditions.14 This beneficial antiadhesive effect of SIN-1 may help to preserve endothelial integrity and function after balloon angioplasty, because adherent neutrophils have been shown to induce endothelial dysfunction.33
The antiadhesive and antithrombotic effects of SIN-1 may be related to inhibition of surface expression of adhesion molecules. Platelet glycoprotein (GP) IIb/IIIa mediates platelet adhesion and aggregation34 and is suggested to be involved in platelet-neutrophil interaction,35 whereas endothelial and platelet P-selectin mediates neutrophil adhesion to endothelium36 and to activated surface-adherent platelets,37 respectively. Langford et al have recently reported that the nitric oxide donor S-nitrosoglutathione prevents platelet activation, as assessed by measurement of GPIIb/IIIa and P-selectin surface expression, during angioplasty38 or unstable angina39 in patients. These findings were corroborated by Michelson et al,40 who found that S-nitroso-N-acetylcysteine inhibits platelet surface expression of GPIIb/IIIa and P-selectin induced by platelet agonists in vitro. Conversely, other studies have shown that inhibition of nitric oxide synthesis induces platelet41 and endothelial42 P-selectin surface expression, which may facilitate neutrophil-endothelium adhesion as well as neutrophil recruitment by activated platelets. These observations support an important regulatory role for nitric oxide in homo- and heterotypic cell interactions and suggest that the ability of SIN-1 to inhibit platelet as well as neutrophil interactions with balloon-injured arteries may be mediated through modulation of the surface expression of adhesion molecules.
The internal diameter of the carotid arteries undergoing angioplasty was similar between the control and SIN-1–treated groups, indicating that SIN-1, at the dose used in our study, had no significant effects on carotid arterial diameter. After balloon dilation, a vasoconstrictive response was observed at the site of endothelial injury induced by the distal tapering end of the balloon.2,3,7-9 This localized vasoconstriction has been reported to be directly related to the extent of platelet adhesion4 and to be reduced by platelet9 or neutrophil7 depletion. Administration of SIN-1 significantly reduced postangioplasty vasoconstriction, which is not surprising given the relaxant properties of SIN-1 on vascular rings or strips without endothelium in vitro.21,43 However, inhibition of platelet and neutrophil-vessel wall interactions may also account for the beneficial effect of SIN-1 on the vascular tone, because the vasomotor response of the balloon-injured artery in vivo is modulated by both platelets4,9 and neutrophils7 and by derived substances. Conversely, this could have limited the vasodilating action of SIN-1 in this model of arterial injury. In this regard, the ability of the cyclooxygenase inhibitor aspirin,9 the dual lipoxygenase/cyclooxygenase inhibitor BW755C,8 and a leukotriene biosynthesis inhibitor (unpublished data, Provost et al. 1996) to prevent postangioplasty vasoconstriction is interesting.
On its decomposition, SIN-1 has been shown to generate superoxide anion, a phenomenon known to occur at concentrations of SIN-1 higher than 300 µmol/L.44 At the dose used in our study, plasma concentration of SIN-1 is estimated to be much lower than that associated with superoxide generation. Although the formation and possible contribution of superoxide anions derived from SIN-1 in vivo seem to be unlikely, the implication of superoxide radicals released by activated neutrophils cannot be excluded. Superoxide anion, which is known to enhance platelet adhesion and aggregation,45 and to contribute to postangioplasty vasoconstriction,46 interacts with nitric oxide to form peroxynitrite, a powerful oxidant with significant cytotoxic potential.47 However, the fate and actions of peroxynitrite in biological systems are such that, in the presence of plasma proteins,48 sugars, or other compounds containing an alcohol functional group,49 it will lead to the formation of compounds with the capacity to generate nitric oxide. There are some pathological conditions in which depletion of thiols may favor the deleterious interaction of peroxynitrite with cell membranes, leading to tissue damage.48 Although a recent study by White et al50 suggests that the reaction of superoxide anion with nitric oxide may be involved in the pathogenesis of atherosclerosis, the potential role of peroxynitrite in the acute and long-term complications of angioplasty injury remains to be defined.
In summary, the efficacy of the nitric oxide donor SIN-1 in
modifying the acute pathophysiological response to
arterial injury by angioplasty in vivo may have important
clinical implications. In vitro, both SIN-151 and
S-nitroso-N-acetylpenicillamine52
have been shown to inhibit vascular smooth muscle cell proliferation.
In a rat carotid artery intimal injury model, Guo et al53
have demonstrated the ability of SPM-5185, a nitric oxide donor, to
inhibit intimal thickening and to accelerate the functional recovery of
the regenerating endothelium. Conversely, chronic
inhibition of nitric oxide production with
N
-nitro-L-arginine methyl ester
impairs endothelium-dependent relaxation and
accelerates neointima formation and
atherosclerosis,54,55 further implicating
nitric oxide in this process. Therefore, administration of a nitric
oxide donor may be beneficial in preventing both the acute and chronic
complications that are associated with arterial injury.
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Acknowledgments |
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SIN-1 was kindly provided by Cassella AG (Frankfurt, Germany). This study was supported by the Heart and Stroke Foundation of Canada (HSFC), and the Fonds de la Recherche en Santé du Québec (FRSQ). Additional support was provided by the Fonds de Recherche de l'Institut de Cardiologie de Montréal. J.T. is the recipient of a grant from the Medical Research Council of Canada and of a senior scholarship from the FRSQ and the HSFC. P.P. was the recipient of a studentship from the Medical Research Council of Canada and his current address is Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77, Stockholm, Sweden. Y.M. is the recipient of a junior scholarship from the FRSQ and the HSFC. We thank Johanne Doucet for excellent technical assistance and Lucie Lacoste for the aggregation studies.
Received July 6, 1995; accepted January 2, 1997.
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