Ann Thorac Surg 2003;76:453-458
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Pressure distension stimulates the expression of endothelial adhesion molecules in the human saphenous vein graft
Massimo Chello, MDa*,
Pasquale Mastroroberto, MDb,
Giacomo Frati, MDa,
Giuseppe Patti, MDa,
Andrea D’Ambrosio, MDa,
Germano Di Sciascio, MDa,
Elvio Covino, MDa
a Department of Cardiovascular Sciences, Interdisciplinary Center for Biomedical Research (CIR), University Campus BioMedico di Roma, Rome, Italy
b Department of Clinical and Experimental Medicine, University of Catanzaro, Catanzaro, Italy
Accepted for publication February 21, 2003.
* Address reprint requests to Dr Chello, Department of Cardiovascular Sciences, Unit of Cardiac Surgery, Università Campus BioMedico di Roma, Via E. Longoni 83, Rome 00155, Italy.
e-mail: m.chello{at}unicampus.it
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Abstract
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BACKGROUND: Mechanical trauma occurring during saphenous vein graft harvesting plays a major role in graft failure after coronary bypass surgery. There is increasing evidence that neutrophil–endothelial interaction is involved in the pathogenesis of early graft occlusion. This study evaluates the effect of pressure distension on the expression of endothelial adhesion molecules in human saphenous vein.
METHODS: Segments of saphenous vein graft (SVG) were collected from 20 patients undergoing coronary bypass surgery. We evaluated the expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and P-selectin on SVG endothelium under basal conditions and after pressure distension at 300 mm Hg. In the same experimental setting we also evaluated adhesion of both unstimulated and activated neutrophils to the endothelium of SVG.
RESULTS: Control endothelial cells exhibited only a weak staining for intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and P-selectin, whereas the levels of adhesion molecules increased significantly in the distended veins. Similarly, significantly greater adhesion of both unstimulated and activated neutrophils was observed in distended veins compared with control veins.
CONCLUSIONS: Pressure distension of SVG before coronary bypass surgery induces upregulation of endothelial adhesion molecules, with subsequent increase in neutrophil adhesion to the endothelium. Neutrophil adhesion to endothelial cells may contribute to early failure of SVG.
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Introduction
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Autologous saphenous vein is still widely used as a graft in coronary artery surgery, although its long-term patency is lower compared with that of arterial conduits [1]. Among the different reasons for short-term failure of saphenous vein graft (SVG), if technical problems are excluded, an important role is played by the neutrophil adhesion to the dysfunctional endothelium [2–5]. In this regard, the mechanical trauma occurring during the SVG harvesting has been widely identified as the major factor responsible for both endothelial dysfunction and anatomical damage [4–8]. In particular, the common practice of injecting saline solution at high pressure to check for leakage from side branches has been found to be associated with endothelial cell injury [9–11]. The neutrophil–endothelial interactions are regulated by the presence of adhesion molecules on the surface of both circulating neutrophils (CD11b/CD18) and endothelial cells (intercellular adhesion molecule [ICAM-1], vascular cell adhesion molecule [VCAM-1, and P-selectin), the expression of which may be modified by a number of factors [12, 13]. Under normal conditions, only a few adhesion molecules are expressed on the endothelium of saphenous vein [14, 15]. However, an upregulation of such molecules has been observed after cytokine stimulation [16] and is influenced by the chemical composition of storage media [6] and by parietal shear stress [17]. The aim of the present study was to evaluate the role of parietal stretching after saline injection on the expression of endothelial adhesion molecules in saphenous vein grafts.
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Patients and methods
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Segments of saphenous vein were obtained from 20 patients undergoing elective coronary artery bypass. There were 16 men and 4 women with an average age of 62 ± 5 years. Criteria for exclusion from the study were diabetes mellitus, peripheral arterial obstructive disease, varicoses, chronic inflammation states, and recent use of steroids. The study was approved by the Ethical Committee of the Medical School of Catanzaro and informed consent was obtained from each patient.
A segment (approximately 10 cm) of freshly isolated vein was taken from the lower portion of the long saphenous vein using routine harvesting technique. After gentle flushing with saline solution to eliminate residual blood, each specimen was divided into two segments of equal length, representing the control and the distended specimen. The latter segments were then subjected to distention for 2 minutes at a pressure of 300 mm Hg by manual injection of saline solution with a syringe connected to a manometer. Injection pressure values were chosen according to those reported to occur during the surgical preparation of the saphenous vein during coronary bypass surgery [11]. Both specimens were then stored in medium 199 with 5% albumin at room temperature for 60 minutes, which is similar to the average storage period for a harvested vein before implantation as a bypass conduit.
Neutrophil isolation
Neutrophils were isolated by Ficoll-Hypaque density gradient centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes. Neutrophils were suspended in Hanks’ balanced salt solution, free of phenol red, Ca 2+, and Mg 2+ and containing 0.25% bovine serum albumin. The final cell preparation had 98% ± 2% neutrophils. The neutrophils were maintained on ice in Hanks’ balanced salt solution at 1 to 5 x10 6 cells/mL until use. Isolated polymorphonuclears (PMNs) were more than 99% pure as assessed by Wrights-stained cytocentrifugation preparation and were more than 99% viable as assessed by exclusion of trypan blue.
Neutrophil activation
Activation of PMNs was performed as previously described [18]. Isolated PMNs were treated with phorbol dibutyrate (3,000 ng/mL) for 15 minutes 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/L; and aprotinin, 0.3 U/mL), and resuspended in medium 199 before the adhesion assay. Treatment of PMNs with phorbol dibutyrate agonist did not alter PMN viability, as judged by exclusion with trypan blue.
Electron microscopy
Scanning electron microscopy was performed to evaluate the damage of the endothelial surfaces of both distended and nondistended saphenous vein. The vein segments were fixed in 3.5% glutaraldehyde in sodium cacodylate buffer. Dehydrated specimens were then dried, sputter coated, and examined with a scanning electron microscope.
Immunohistochemistry
Immunostaining was used to investigate the distribution of the endothelial adhesion molecule in segments of human saphenous vein. Saphenous vein was dehydrated using graded acetone washes and was embedded at 4°C. Sections 6 µm thick were cut and transferred to coated slides (Vectabond; Vector Laboratories, Burlingham, CA). Immunohistochemical localization of P-selectin, ICAM-1, and VCAM-1 was achieved using the avidin-biotin immunoperoxidase technique as described by Chester and coworkers [14]. Tissue sections were incubated with one of the monoclonal primary antibodies (Research Diagnostic, Flanders NJ at 1:100 dilution) overnight at room temperature. Biotynilated immunoglobulin G (IgG) was used as the secondary antibody at a 1:200 dilution for 1 hour at room temperature. The avidin-biotin immunoperoxidase technique was used to detect biotinylated secondary antibody. Immunostaining negative controls were performed by omitting the primary antibody or secondary antibody. The expression of adhesion molecules was interpreted according to Lefer and coworkers [19], as the percentage of examined vein segment displaying brown staining on more than 50% of the circumference of its endothelium.
Polymorphonuclear adherence assay
Segments of saphenous vein from both distended and control groups were opened carefully and placed endothelial side up in separate 5-mL round cell culture dishes containing 3 mL of Krebs-Henseleit (Fluka, Sigma-Aldrich, Milan, Italy) solution, according to Ma and coworkers [20]. Experiments were performed by placing the cell dishes in a Plexiglas chamber at a constant temperature (37°C) and normoxic atmosphere (21% O2, 5% CO2, 74% N2) .
The PMNs were labeled with a hydrophobic fluorescent compound (3 to 3'-Dioctadecyloxacarbocyanine perchlorate (DiI) (Fluka, Sigma-Aldrich, Milan, Italy) as previously described [21]. Cells at 4 to 8 x 106 cells/mL were incubated with 50 µg/mL DiI in HAP buffer for 10 minutes 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. After 10 minutes of preincubation of the vessel segments, autologous unstimulated DiI-labeled PMNs (10 µL of 106 cells/mL) were added and incubated for 20 minutes. Vessel 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 and were covered with a thin glass slide. 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 to 550. Values of five replicates were averaged, and variations between replicates were less than 10%.
In a subset of experiments (N = 10), the importance of endothelial circulating molecules in determining PMN/endothelial adhesion was evaluated by treating saphenous vein endothelium with blocking monoclonal antibodies (mAb) to P-selectin, ICAM-1, and VCAM-1.
Statistical analysis
All values are expressed as mean ± SEM. Comparison 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 less than 0.05
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Results
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Endothelial coverage
Scanning electron microscopy of nondistended saphenous vein showed almost no damage to the endothelial layer. Analysis of the distended grafts showed numerous areas of endothelial denudation and focal sites of partially detached cells; the mean endothelial loss was 33% ± 2.2% (Fig 1).
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Fig 1. (A) Scanning electron microscopy of control vein with almost intact endothelial layer (N = nucleus). Areas of endothelial denudation (arrow) with partly detached cells are present after pressure distension (C).
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Expression of endothelial adhesion molecules
Figure 2
represents the percentage of vein segments with positive staining for VCAM-1, ICAM-1, and P-selectin in both the control and distended vein grafts. We found that in the control vein segments, ICAM-1 (Fig 3a)
and P-selectin (Fig 4a)
staining was only weakly positive on the endothelial cells, whereas VCAM-1 staining was almost negligible. By contrast, in the distended vein grafts, the staining intensity of adhesion molecules was markedly increased compared to control vein segments (Figs 3b, 4b). In the case of P-selectin, it was observed that the percentage of cells that exhibited enhanced expression was 21.3% ± 1.5% (p < 0.01 vs control, 95% CI = 6.13 to 11.07). For VCAM-1 and ICAM-1, the percentages were 5.8% ± 0.3% (p < 0.01 vs control 95% CI = 2.45 to 4.15) and 11.4% ± 1.1% (p < 0.01 vs control, 95% CI = 13.46 to 19.24), respectively.
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Fig 2. Percent expression of intercellular adhesion molecule–1 (ICAM-1), vascular cell adhesion molecule–1 (VCAM-1), and P-selectin on the endothelial cells of control and dilated saphenous vein grafts. Bars represent mean number (± SEM) of positive staining segments per area examined, with filled bars indicating control and open bars dilated saphenous vein grafts. (*p < 0.01 versus control vein.)
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Fig 3. Immunohistochemical analysis of intercellular adhesion molecule-1 expression on the endothelium of human saphenous vein. Staining was negative on endothelial cells of control vein (a), but it increased significantly (arrows) on the endothelial cells of distended vein (b).
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Fig 4. Immunohistochemical analysis of P-selectin expression on endothelium of human saphenous vein. Staining was only weakly positive on endothelial cells of control veins (a), but it increased significantly (arrows) on endothelial cells of distended veins (b).
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Pmn adhesion to SV endothelial cells
Figure 5
shows the percentage values of neutrophil adhesion to the endothelium of SVG segments. Under base line conditions, very few PMNs bound to endothelium of control veins; after activation of PMNs, the adhesion and ruffle formation of neutrophils on the endothelium of control veins was markedly enhanced (p < 0.01, 95% CI = 6.66 to 11.64 vs nonactivated neutrophils). In the distended veins the percentage values for the number of nonactivated neutrophils adhering to the endothelial cells was significantly higher than in the control group (p < 0.01, 95% CI = 4.62 to 8.68). This difference was even more pronounced when segments of the distended veins were incubated with activated neutrophils (control 11.3 ± 0.9%, distended 20.1% ± 1.5%, p < 0.01 95% CI = 1.78 to 9.71). Figure 5 also shows the results of a subset of experiments in which the dilated vein segments (N = 10) were incubated with the following blocking monoclonal antibodies: to P-selectin (1:200 dilution), to ICAM-1 (1:100 dilution), and to VCAM-1 (1:100 dilution). The blocking of VCAM-1 was accompanied by a fair but not significant decrease in PMN adhesion (-1.5%, p = ns vs no blocking mAb), whereas a significant reduction of neutrophil adhesion to the endothelium was observed after the blocking P-selectin (-6.3% p < 0.01 vs no blocking mAb), ICAM-1 (-4.6%, p < 0.05 vs no blocking mAb), or ICAM-1 plus P-selectin (-12.5%, p < 0.01 vs no blocking mAb)
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Fig 5. Adhesion of both unstimulated and activated polymorphonuclears (PMNs) to endothelium of either dilated or control veins, under normal conditions and after treatment with blocking monoclonal antibodies (mAb) to vascular cell adhesion molecule–1 (VCAM-1), intercellular adhesion molecule–1 (ICAM-1), P-selectin (P-sel), and P-selectin plus ICAM-1. Bars indicate mean values ± SEM, with filled bars representing control and open bars dilated veins. (*p < 0.01 versus control vein.)
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Conclusions
Leukocyte adhesion to the vascular endothelium, which represents an important step in the early stages of venous graft obstruction, is mediated by the interaction of adhesion molecules expressed on the surfaces of both endothelial cells and leukocytes. The substances ICAM-1, VCAM-1, and P-selectin mediate the binding of leukocytes to endothelial cells through interactions with their counter-receptors on leukocytes [12, 13]. It has been demonstrated that the expression of these adhesion molecules is very low on segments of saphenous vein that have been harvested at the time of operation before coronary grafting [14, 15], but they can be up-regulated by a number of different mechanical and chemical stimuli [3, 6, 16, 17].
In this study, we have examined the effects of internal pressurization on the expression of endothelial adhesion molecules in human saphenous vein grafts. By comparing matched pairs of vein tissue we demonstrated that a 2-minutes period of distension at 300 mm Hg results in an increased expression of the endothelial adhesion molecules. This in turn determines a significant increase in neutrophil adhesion to the endothelium of the vascular graft, as demonstrated by experiments performed with the blocking monoclonal antibodies to both P-selectin and ICAM-1.
The substance VCAM-1, which is usually almost absent on unstimulated endothelial cells, is known to mediate the adhesion of lymphocytes and monocytes in inflamed vascular beds [12]. In the present study, VCAM-1 exhibited a weaker upregulation compared with ICAM-1, which is expressed at basal levels on the endothelial cells. P-selectin, which showed the highest percentage value of upregulation, is an endothelial adhesion molecule that is constitutively expressed in a preformed state in the 
granules of platelets and in the Wiebel-Palade bodies of endothelial cells [21]. It is rapidly translocated to the cell surface after stimulation with inflammatory mediators, allowing rolling and adhesion of activated PMNs to the endothelium [22]. The latter event is crucial in mediating leukocyte adhesion to platelets and to the endothelium after ischemia reperfusion. The effect of different types of mechanical stimuli on the expression of adhesion molecules in SVG has been the subject of previous studies. Chappell and coworkers [17] have shown that oscillatory flow imposed in an in vitro environment has the capacity to induce the expression of adhesion molecule on human cultured umbilical vein cells. Their results suggest that the areas of the endothelium that have oscillatory flow in vivo may express enhanced levels of surface VCAM-1, ICAM-1, and E-selectin.
Golledge and coworkers [23], using an in vitro model of human saphenous vein bypass, reported a twofold increase in ICAM-1 expression in unstented saphenous vein exposed to arterial flow, which could be prevented by limiting the circumferential deformation of the vein with an external polytetrafluoroethylene stent.
Endothelial nitric oxide (NO) is fundamental to vascular function. Nitric oxide, which is produced from L-arginine, not only regulates vascular tone but has been shown to modulate significantly the leukocyte–endothelial cell interaction by suppressing the up-regulation of several endothelial cells and PMN adhesion molecules, including P-selectin, VCAM-1, and CD11b/CD [19, 24]. In a previous study we demonstrated that neutrophil–endothelial interactions are higher in the SVGs compared with the internal mammary artery, as a consequence of the reduced NO production by the venous endothelium [21].
Therefore, it could be speculated that the reduced bioavailability of NO might cause the up-regulation of endothelial adhesion molecules observed in the dilated vein grafts. Several recent studies support the view that reduced expression of endothelial nitric oxide synthase (eNOS), and the consequent lower NO production, may be caused by the endothelial loss during the harvesting process. Tsui and coworkers [25] demonstrated a reduced expression of endothelial nitric oxide (eNO) synthase in the endothelium of saphenous veins harvested according to standard technique compared with saphenous vein grafts harvested with a nontouch technique. Chester and coworkers reported that segments of SVG obtained during CABG operation and injected at a 300 mm Hg pressure exhibited a reduced response to the endothelium-dependent dilatory effects of acetylcholine [9]. These investigators attributed this finding to a reduced bioavailability of NO synthase in connection with areas of de-endothelization observed in this model of venous graft. Finally, Liu and coworkers [26] reported a reduced basal release of NO in the SVG subjected to mechanical distension compared with the control veins. Furthermore, the maximum concentrations of NO release induced by acetylcholine in the distended grafts were also significantly lower than those in the control veins.
The presence of areas of endothelial loss could also increase the chance of adhesion of activated neutrophils and platelets to the subendothelium, as demonstrated by Angelini and coworkers [10]. These investigators, using a porcine model of autologous saphenous vein to common carotid artery bypass grafting, reported an extensive reduction of endothelial cover (98% vs 38% of nondistended veins) in saphenous vein grafts undergoing mechanical distension at 600 mm Hg, which was associated with increased platelet and leukocyte adhesion and with reduced early graft patency. In our study, the areas of endothelial loss (mean 33%) observed in the distended veins are lower than those reported by Angelini and coworkers and closer to those in other studies using human saphenous vein and lower injection pressures [7, 8, 26]. In addition, in contrast to other studies, we stored the segments of SVG in medium 199 with albumin, which has been shown to be superior to physiologic saline in preserving endothelial cells [8]. Finally, the dramatic drop of neutrophil–endothelial adhesion after treatment with mAb to ICAM-1 and P-selectin clearly identify these molecules as the main factors responsible for the observed difference in neutrophil–endothelial interactions between dilated and control grafts.
In conclusion, mechanical distension of SVG up-regulates endothelial adhesion molecules, which consequently increase neutrophil–endothelial cell adhesion. Neutrophil adhesion represents an early step in SVG occlusion. For this reason, efforts should be made to avoid unnecessary mechanical distension of the saphenous vein at the time of coronary artery bypass surgery.
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References
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- Cooper G.J., Underwood M.G., Deverall P.B. Arterial and venous conduits for coronary artery bypass. A current review. Eur J Cardiothorac Surg 1996;10:129-140.[Abstract]
- Bryan A.J., Angelini G.D. The biology of saphenous-vein graft occlusion: etiology and strategies for prevention. Curr Opin Cardiol 1994;9:641-649.[Medline]
- Thatte H.S., Khuri S.F. The coronary artery bypass conduit: I. Intraoperative endothelial injury and its implication on graft patency. Ann Thorac Surg 2001;72:S2245-S2252.[Abstract/Free Full Text]
- Motwani J.G., Topol E.J. Aortocoronary saphenous vein graft diseases: pathogenesis, predisposition and prevention. Circulation 1998;97:916-931.[Abstract/Free Full Text]
- Mills N.L., Everson C.T. Vein graft failure. Curr Opin Cardiol 1995;10:562-568.[Medline]
- Schaeffer U., Tanner B., Strohschneider T., Stadmuller A., Hannekum A. Damage to arterial and venous endothelial cells in bypass grafts induced by several solution used in bypass surgery. Thorac Cardiovasc Surg 1997;45:168-171.[Medline]
- Alrawi S.J., Balaya F., Raju R., Cunningham J.N., Jr, Acinapura A.J. A comparative study of endothelial cell injury during open and endoscopic saphenectomy: an electron microscopic evaluation. Heart Surg Forum 2001;4:120-127.[Medline]
- Hickethier T., Dammrich J., Silber R.E., Finster S., Elert O. Ultrastructural investigations for reducing endothelial cell damage of vein grafts during CABG-operation and practical consequences. J Cardiovasc Surg (Torino) 1999;40:71-76.[Medline]
- Chester A.H., Buttery L.D., Borland J.A., et al. Structural, biochemical, and functional effects of distending pressure in the human saphenous vein implications for bypass grafting. Coron Artery Dis 1998;9:143-151.[Medline]
- Angelini G.D., Bryan A.J., Williams H.M., Morgan R., Newby A.C. Distention promotes platelet and leukocyte adhesion and reduces short-term patency in pig arteriovenous bypass grafts. J Thorac Cardiovasc Surg 1990;99:433-439.[Abstract]
- Galea J., Armstrong J., Francis S.E., Cooper G., Crossman D.C., Holt C.M. Alterations in c-fos expression, cell proliferation and apoptosis in pressure distended human saphenous vein. Cardiovasc Res 1999;44:436-448.[Abstract/Free Full Text]
- Krieglstein C.F., Granger D.N. Adhesion molecules and their role in vascular disease. Am J Hypertens 2001;14:44S-54S.[Medline]
- Zilla P., Oppell U., Deutsch M. The endothelium: a key to the future. J Card Surg 1993;8:32-60.[Medline]
- Chester A.H., Morrison K.J.M., Yacoub M.H. Expression of vascular adhesion molecules in saphenous vein coronary bypass. Ann Thorac Surg 1998;65:1685-1689.[Abstract/Free Full Text]
- Chester A.H., Borland J.A., Taylor P.M., Rose M.I., Yacoub M.H. Vascular adhesion molecules and immunogenicity in blood vessels used as coronary artery bypass grafts. Eur J Cardiothorac Surg 1996;10:676-683.[Abstract]
- Crook M.F., Newby A.C., Southgate K.M. Expression of intercellular adhesion molecules in human saphenous veins: effects proinflammatory cytokines and neointima formation in culture. Ahterosclerosis 2000;150:33-41.
- Chappell D.C., Varner S.E., Nerem R.M., Medford R.M., Alexander R.W. Oscillatory shear stress stimulates adhesion molecules expression in cultured human endothelium. Circ Res 1998;82:532-539.[Abstract/Free Full Text]
- Chello M., Mastroroberto F., Perticone F., Celi V., Colonna A. Nitric oxide modulation of neutrophil-endothelium interaction: difference between arterial and venous coronary bypass grafts. J Am Coll Cardiol 1998;31:823-826.[Abstract/Free Full Text]
- Lefer A.M., Campbell B., Shin Y.K., Scalia R., Hayward R., Lefer D.J. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation 1999;100:178-184.[Abstract/Free Full Text]
- Ma X., Weyrich A.S., Lefer D.J. Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium. Circ Res 1993;77:403-412.
- McEver R.P., Beckstead J.H., Moore K.L., Marshall-Carlson L., Bainton D.F. GMP-140 a platelet alpha-granule membrane protein is also synthesized by vascular endothelial cells is localized in Weibel-Palade bodies. J Clin Invest 1989;84:92-99.
- Geng J.G., Bevilacqua M.P., Moore K.L., et al. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature 1990;343:757-760.[Medline]
- Golledge J., Tumer R.J., Harley S.L., Springall D.R., Powell J.T. Development of an in vitro model to study the response of saphenous vein endothelium to pulsatile arterial flow and circumferential deformation. Eur J Vasc Endovasc Surg 1997;13:605-612.[Medline]
- Kubes P., Suzuki M., Granger D. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991;88:4651-4655.[Abstract/Free Full Text]
- Tsui J.C., Souza D.S., Filbey D., Bomfim V., Dashwood M.R. Preserved endothelial integrity and nitric oxide synthase in saphenous vein grafts harvested by a ‘no-touch’ technique. Br J Surg 2001;88:1209-1215.[Medline]
- Liu Z.G., Liu X.C., Yim A.P., He G.W. Direct measurement of nitric oxide release from saphenous vein: abolishment by surgical preparation. Ann Thorac Surg 2001;71:133-137.[Abstract/Free Full Text]
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Abstract
Introduction
