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JACC Vol. 31, No. 4
March 15, 1998:823– 6
Nitric Oxide Modulation of Neutrophil-Endothelium Interaction:
Difference Between Arterial and Venous Coronary Bypass Grafts
MASSIMO CHELLO, MD, PASQUALE MASTROROBERTO, MD, FRANCESCO PERTICONE, MD,
VITTORIA CELI, MD, ALFREDO COLONNA, PHD*
Catanzaro and Naples, Italy
Objectives. This study sought to evaluate the relation between
the pattern of neutrophil-endothelial adhesion in saphenous vein
(SV) and internal mammary artery (IMA) grafts and the endothelial production of nitric oxide (NO).
Background. Autologous IMA and SV grafts (SVGs) are increasingly used as conduits for coronary bypass grafting. Previous
studies have demonstrated a greater production of endothelialderived relaxing factor (NO) from IMA than from SVGs. Because
of the well known role of NO in modulating the adhesion of
polymorphonuclear leukocytes to the endothelium, we studied the
pattern of neutrophil adhesion to the endothelium of IMA and
SVs under basal conditions and after inhibition of NO synthesis.
Methods. Segments of IMA and SVs were obtained from 20
patients undergoing coronary artery bypass graft surgery. We
evaluated the adhesion of both unstimulated and activated neutrophils to the endothelial surface of IMA and SVs in both basal
conditions and after inhibition of NO synthesis with Nv-nitro-Larginine methyl ester.
Results. Under basal conditions, no difference in unstimulated
neutrophil adhesion to endothelium was observed between the two
vessel conduits. After neutrophil activation, a significantly (p <
0.05) greater adhesion of neutrophils was observed in the SV than
in the IMA. After inhibition of NO release, the adhesion of
activated neutrophils increased in both vessels, and no significant
difference between them was observed. The increased adhesion
was attenuated by both L-arginine and sodium nitroprusside.
Conclusions. The lesser neutrophil adhesion to the endothelium
of the IMA is a consequence of enhanced release of NO at this
level; this effect could be responsible for the better early and
long-term patency of this conduit over the SVG in coronary bypass
grafting.
(J Am Coll Cardiol 1998;31:823– 6)
©1998 by the American College of Cardiology
The two most commonly used conduits for coronary artery
bypass grafting are the autologous internal mammary artery
(IMA) graft and the saphenous vein graft (SVG), with increasing use of the IMA because of its superior early and long-term
patency (1). The reason for the greater long-term durability of
the IMA graft remains obscure, although numerous studies
(2,3) have investigated the histologic differences between these
two types of conduit materials as well as the differences in their
biochemical composition. There is increasing evidence (4,5)
that endothelium-blood cell interaction plays an important
role in the pathogenesis of both early and late graft occlusions.
Although the role of platelets in this context has largely been
proved by experimental and clinical studies, the link between
polymorphonuclear leukocytes (PMNs) and atherosclerosis
has only recently been ascertained. Epidemiologic evidence (6)
shows that neutrophil count is a risk factor in predicting
occurrence of cardiovascular or cerebrovascular accident, although a causative mechanism has not been established. It is
now well recognized (7,8) that PMNs significantly mediate
both coronary and pulmonary endothelium injury associated
with ischemia and reperfusion. Furthermore, several in vitro
studies (9) have shown that activated PMNs injure endothelial
cell monolayers. Neutrophil adherence probably contributes to
endothelial injury by the release of reactive oxygen species and
proteolytic enzymes (8,9).
The endothelium modulates the responsiveness on the
underlying vascular smooth muscle mostly by the release of
potent relaxing factors (10,11), one of which has been identified as nitric oxide (NO) or as a donor of NO, which is
produced from L-arginine (12,13). NO, produced by endothelial cells, not only regulates vascular tone, but was recently
shown (14) to play a significant role in leukocyte-endothelial
interaction. Reduced endothelial NO production results in
increased neutrophil adherence and enhanced microvascular
permeability in the postcapillary venule (15); moreover, inhibition of endogenous NO formation results in pronounced
increase of PMN adherence to endothelium through upregulation of the CD11b/CD18 adhesion glycoprotein (14).
The purposes of the present study were to determine the
From the Department of Experimental and Clinical Medicine, Divisions of
Cardiac Surgery, Internal Medicine and Biochemistry, Medical School of Catanzaro, Catanzaro; and *Division of Biochemistry, Faculty of Pharmacy, University
Federico II, Naples, Italy. This work was supported in part by a grant from the
Italian Ministry of University and Scientific Research, Rome, Italy.
Manuscript received April 28, 1997; revised manuscript received December
4, 1997, accepted December 12, 1997.
Address for correspondence: Dr. Massimo Chello, Via San Giacomo dei
Capri 29, 80128 Naples, Italy. E-mail: [email protected].
©1998 by the American College of Cardiology
Published by Elsevier Science Inc.
0735-1097/98/$19.00
PII S0735-1097(97)00560-3
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CHELLO ET AL.
NITRIC OXIDE AND NEUTROPHIL ADHESION
JACC Vol. 31, No. 4
March 15, 1998:823– 6
Abbreviations and Acronyms
DiI
5 dioctadecyloxacarbocyanine perchlorate
EDRF 5 endothelium-derived relaxing factor
IMA
5 internal mammary artery
K-H
5 Krebs-Henseleit
v
L-NAME 5 N -nitro-L-arginine methyl ester
NO
5 nitric oxide
PDB
5 phorbol dibutyrate
PMN
5 polymorphonuclear leukocyte
SV
5 saphenous vein
SVG
5 saphenous vein graft
pattern of PMN adhesion to the endothelium of both IMA and
SVGs and to relate basal endothelium release of NO to
alterations of PMN adherence to vascular endothelium.
Methods
Material. The material for this study was obtained from 20
patients undergoing coronary artery bypass surgery, in whom
the IMA and the saphenous vein (SV) were used as bypass
grafts. There were 15 men and 5 women with a mean age of
54.5 years (range 41 to 68). Written informed consent was
obtained from each patient. The study was approved by the
Ethical Committee of the Medical School of Catanzaro.
Neutrophil isolation. Human neutrophils were purified
from patient blood on Ficoll-sodium diatrizoate (Hypaque)
gradients. Isolated PMNs were .99% pure as assessed by
Wright’s stained cytocentrifuge preparation and .99% viable
as assessed by exclusion of trypan blue.
PMN activation. PMN activation was performed according
to the method of Lo and co-workers (16). Labeled PMNs were
treated with phorbol dibutyrate (PDB, 300 ng/ml) for 15 min
at 37°C, washed three times with HAP buffer (Dulbecco’s
phosphate buffered saline containing human serum albumin,
0.5 mg/ml; glucose, 3 mmol/liter; and aprotinin, 0.3 U/ml), and
suspended in medium 199 before the adhesion assay. Treatment of PMNs with this agonist did not alter PMN viability, as
judged by exclusion with trypan blue.
PMN adherence assay. PMNs were fluorescently labeled
with a hydrophobic fluorescent compound (3-39-dioctadecyloxacarbocyanine perchlorate (DiI) (Fluka, Sigma-Aldrich, Milan, Italy) as described by Lo et al. (16). Cells at 4 to 8 3 106
cells/ml were incubated with 50 mg/ml DiI in HAP buffer for
10 min at 0°C, unbound dye was removed by three washes with
HAP buffer and labeled PMNs were resuspended in medium
199 for the adhesion assay.
Segments of both IMA and SV harvested at the time of
bypass surgery were opened carefully and placed endothelial
side up in separate 5-ml round cell-culture dishes containing 3
ml of Krebs-Henseleit (K-H) solution, as described by Ma and
coworkers (17). After 10 min of preincubation of the vessel
segments, autologous unstimulated DiI-labeled PMNs (10 ml
of 106 cells/ml) were added and incubated for 20 min. Vessel
Figure 1. Bar graph showing leukocyte adhesion to endothelium of
IMA artery and SVG under basal conditions, after neutrophil activation by PDB and after addition of L-NAME, L-NAME plus L-arginine
(L-Arg), and L-NAME plus nitroprusside (Nipride) to the tissue bath.
Heights of the bars indicate mean values. *p , 0.05 IMA versus SVG.
segments were then removed from culture dishes and dipped
three or four times in fresh K-H solution. These vessel
segments were then placed on a glass slide with the endothelial
side up. The number of PMNs adhering to the endothelial
surface in five separate microscopic fields was counted manually on an inverted microscope equipped for fluorescence using
the filter IF355-550. Values of five replicates were averaged,
and variations between replicates were ,10%.
In a second series of experiments, PDB-stimulated autologous PMNs were incubated for 20 min with both SV and IMA
vessel segments. PMN adhesion to the endothelium was evaluated as previously described.
Inhibition of basal NO release from IMA and SV endothelial cells. In the present study, we observed the effect of
inhibiting basal NO release from both the IMA and the SV on
PMN adherence to endothelium. Nv-nitro-L-arginine methyl
ester (L-NAME, 1 mmol/liter) was incubated with vessel
segments for 30 min. These vessel segments were then transferred to fresh K-H solution that did not contain L-NAME.
Stimulated autologous PMNs were then incubated for another
20 min with these vessel segments. PMN adhesion to the
endothelium was evaluated as described before.
In other experiments, the L-NAME vessel segments were
coincubated with L-arginine (1022 mol/liter) or nitroprusside
(1 mmol/liter) to determine whether substrate or exogenous
NO inhibits neutrophil adhesion to L-NAME–treated vascular
grafts.
Statistics. All values are expressed as mean value 6 SEM.
Comparisons between groups were made by using two-way
analysis of variance followed by the Bonferroni correction for
t test comparison. Statistical significance was set at p , 0.05.
Results
PMN adhesion. Figure 1 shows the percent of neutrophil
adhesion to the endothelium of SVG and IMA segments.
Without stimulation, very few PMNs bound to endothelium,
JACC Vol. 31, No. 4
March 15, 1998:823– 6
CHELLO ET AL.
NITRIC OXIDE AND NEUTROPHIL ADHESION
and no significant difference was observed between the two
vessels. In contrast, when PMNs were stimulated by PDB
treatment, the adhesion and ruffle formation of neutrophils on
endothelium were markedly enhanced in both vessel segments.
However, on the basis of counts made before and after
washing, the percent of PMNs adhering to the IMA endothelium was significantly lower than that in the SVG endothelium
(p , 0.05).
In an attempt to relate the reduced PMN adherence to the
IMA endothelium to the greater basal NO production, we
studied the effects of L-NAME added directly to the bath.
Figure 1 summarizes the results. Addition of L-NAME directly
to the bath significantly increased PMN adherence in both
vessel segments, with a greater increment observed in the
IMA. Although the percent of neutrophils adhering to the
IMA endothelium was lower than that observed in the SVG
endothelium, this difference failed to reach statistical significance. Moreover, Figure 1 shows that both the addition of
L-arginine and the supplementation of exogenous NO by
nitroprusside significantly reduced L-NAME–induced neutrophil adhesion to vascular endothelium. These results suggest
that endogenous NO generated from IMA endothelium acts as
an inhibitor of neutrophil adherence.
Discussion
The aortocoronary bypass operation has been widely applied to the treatment of ischemic heart disease. There is
uniform agreement that, if technical problems are excluded
(18,19), early graft failure is mainly linked to acute thrombosis,
whereas late graft failure is mostly due to atherosclerotic
changes within the vessel wall (20).
Neutrophils and graft occlusion. Although the pathogenetic mechanisms that lead to early and late graft occlusion are
not completely understood, evidence continues to grow for the
important role of neutrophil-endothelial interactions, regulated by both humoral and local mediators. Although the exact
pathophysiology of the damage remains unclear, the presence
of leukocytes in the vessel wall through the release of proteolytic enzymes and the generation of oxygen free radicals may
aggravate the endothelial damage and further stimulate platelets, thus having a potential bearing on the subsequent development of restenosis (21). Conversely, activated granulocytes
have been shown (22) to increase the response of platelets to
stimulation and directly to cause platelet aggregation. Platelets
can also adhere directly to granulocytes, so that deposition or
activation of platelets in ischemic tissue could promote local
neutrophil accumulation. Boogaerts et al. (23) reported that
increased PMN aggregation and adherence induced by
platelet-derived products augmented endothelial cell damage,
probably by increasing expression of complement receptors on
human PMNs. Angelini et al. (24) using a pig model of
autologous SV to common carotid artery bypass grafting
found an extensive leukocyte and platelet adhesion in deendothelialized area of vein grafts, especially in those made
with distended veins. Likewise, platelet and leukocyte adhesion
825
has been observed in human SVs examined shortly after
grafting in the coronary circulation (18,19).
Boerboom et al. (25) evaluated the histologic and morphometric evolution of vein grafts in a nonhuman primate model.
They found a significant increase in the prevalence of PMNs on
the lumen surface and in both the intima and the media during
the first 14 days after grafting. A leukocyte invasion into grafts
during the first week after implantation has also been described, and the selective invasion of these cells has been
regarded (26) as a response triggered by wall ischemia, fragmentation or necrosis of myocytes, and hemorrhagic foci.
Neutrophils could also play a role in the formation of the
atheromatous plaque. In experimental models of atherosclerosis (27), neutrophils infiltrate damaged endothelium, and
these cells have the potential to modulate endothelial permeability and cause local tissue damage and proliferation. As
suggested by Nash and Shearman (28), it might be postulated
that increased circulating levels of chemoactractans or cytokines would cause chronic or periodic neutrophil stimulation,
thus promoting formation of atheroma.
Although passive neutrophils circulate in a nonadhesive
state, they can at any time become adhesive. Ricevuti (29)
demonstrated that PMN aggregability in the coronary sinus is
greater in patients with angiographically documented coronary
disease than in control subjects with normal coronary vessels.
An important first step in the process of neutrophil-mediated
endothelial damage involves the binding of neutrophils to
endothelial cells. This process is largely regulated by complementary adhesion molecules; some of these are present essentially on the cell surface and others can be up-regulated in
response to chemotactic and proinflammatory stimuli.
NO modulation of PMN-endothelium interaction. Recently NO has been shown to be an endogenous inhibitor of
leukocyte chemotaxis adherence (14) and activation (30).
Inhibition of NO synthesis resulted in increased leukocyte
adherence in postcapillary venules, a response that could be
prevented by adding exogenous NO (15) or a high concentration of L-arginine (14).
The results of the present study confirm the important role
that NO plays in the modulation of neutrophil-endothelium
interactions. The addition of L-NAME, a potent inhibitor of
endothelial NO synthesis, to the endothelium of both SV and
IMA grafts significantly increased neutrophil adherence to
endothelial cells, with an almost similar percent of adherent
PMNs observed in SV and IMA grafts. Moreover, this increased adhesion was prevented by both L-arginine and the NO
donor nitroprusside. Thus, it is possible to speculate that the
significantly lower PMN adherence to the endothelium of the
IMA observed under basal conditions is a consequence of a
greater production of endothelium-derived NO in the IMA
than in the SVG. These results are in agreement with those of
recent studies (31,32) using human models. The hypothesis
that the superior patency of the IMA graft could be due to
enhanced production of endothelium-derived relaxing factor
(EDRF) was first made by Luscher and co-workers (33), who
demonstrated that endothelium-dependent relaxation in re-
826
CHELLO ET AL.
NITRIC OXIDE AND NEUTROPHIL ADHESION
sponse to acetylcholine and platelet-derived products was
greater in the IMA than in the SV, both obtained from patients
undergoing coronary bypass surgery. In a similar fashion,
Pearson and co-workers (34), using an experimental model of
canine IMA perfusion, demonstrated that the right and the left
IMA exhibited a significant intraluminal and extraluminal
release of EDRF. Conversely, Chua et al. (35) observed a small
basal release of EDRF from human SVs removed from
patients undergoing elective coronary bypass surgery. They
showed that the endothelium-dependent relaxation elicited by
acetylcholine was impaired markedly in SVs grafted into the
arterial circulation.
Conclusions. The results of our study demonstrate that the
reduced production of endothelium-derived NO in the SVG
compared with that in the IMA results in a greater neutrophilendothelial adhesion. These findings suggest one possible
mechanism for the observed net difference between the two
types of conduits in early and long-term patency rates.
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