Ann Thorac Surg 2006;81:1262-1268
© 2006 The Society of Thoracic Surgeons
Original article: Cardiovascular
Neutrophil Adherence to Activated Saphenous Vein and Mammary Endothelium After Graft Preparation
Axel Schlitt, MD
a
,
*
,
Diethard Pruefer, MD
b
,
*
,
Ute Buerke, MD
a
,
Martin Russ, MD
a
,
Manfred Dahm, MD
b
,
Hellmut Oelert, MD
b
,
Karl Werdan, MD
a
,
Michael Buerke, MD
a
,
*
a Department of Medicine III, Martin Luther-University, Halle-Wittenberg, Germany
b Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University, Mainz, Germany
Accepted for publication September 15, 2005.
* Address correspondence to Dr Buerke, Department of Medicine III, Martin Luther-University, Ernst-Grube-Strasse 40, 06097 Halle/Saale, Germany (Email: michael.buerke{at}medizin.uni-halle.de).
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Abstract
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BACKGROUND: Interaction of circulating leukocytes and vascular endothelium plays an important role in vasoconstriction, endothelial dysfunction, and vascular injury. Dilation procedures of grafts before coronary artery bypass graft surgery might lead to vascular injury and subsequent bypass graft disease.
METHODS: We analyzed in vitro the adherence of fluorescence-labeled polymorphonuclear neutrophils (PMNs) to endothelium of human saphenous vein grafts or internal mammary artery grafts after stimulation with thrombin (0.5 to 2 U/mL) or dilating procedures. Furthermore, we investigated endothelial function of prepared grafts.
RESULTS: Thrombin stimulation resulted in a dose-dependent increase of PMN adherence to the endothelium of saphenous vein and internal mammary artery, which was attenuated by the selectin-blocking carbohydrate fucoidin or anti–P-selectin monoclonal antibody. Mechanical dilation of saphenous vein or internal mammary artery led to a marked increase in PMN adherence (65 ± 5 versus 5 ± 3 PMN/mm2; p < 0.01), which was significantly attenuated by fucoidin or anti–P-selectin monoclonal antibodies. Treatment of internal mammary artery with the vasodilator papaverine led to a marked increase of PMN adherence (59 ± 8 versus 12 ± 4 PMN/mm2; p < 0.01) when papaverine was administered directly into the vessel. However, external treatment with papaverine did not affect PMN adhesion. Endothelial dysfunction was observed in dilated venous grafts and in arterial grafts internally treated with papaverine; in contrast, external treatment did not affect endothelial function.
CONCLUSIONS: This study showed that mechanical or pharmacologic dilation of venous or arterial coronary grafts, usually performed before anastomosis of aortocoronary bypass grafts, led to increased selectin-mediated PMN adhesion on vascular endothelium and subsequent endothelial dysfunction.
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Introduction
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Despite the use and superior patency rate of the internal mammary artery (IMA) and other arterial conduits, saphenous vein grafts (SV) continue to be the most commonly used conduits for coronary artery bypass graft surgery. However, the long-term patency of SV is low compared with arterial grafts. The occlusion rate in the first year is 15% to 26% [1, 2]. By 10 to 12 years, approximately 50% of SV are occluded, and of those still patent, 50% show marked atherosclerotic lesions. Morphologic examination of venous grafts suggests three different modes of occlusion [1]. First, early after operation (ie, within 1 month) occlusion is usually thrombosis related; second, within the first year, graft occlusion is caused by intimal proliferation and medial fibrosis; and third, after 1 year, atherosclerosis is the most common cause of graft occlusion [1].
Recently, it has been shown that the use of high-pressure distention to overcome vasospasm during graft harvesting causes endothelial cell loss and medial damage [3]. Endothelial cell loss denudes the surface of the intima, causing deposition of platelets and fibrin as well as intimal hyperplasia [4, 5]. In addition to the mechanical dilation procedure, a variety of pharmacologic agents have been tested for their ability to prevent venous or arterial spasms. Papaverine is a commonly used vasodilator to reduce vasospasm. However, there is evidence that papaverine may damage the endothelium owing to its acidic pH and may reduce prostacyclin production in the vascular wall [6].
The main purposes of the present study were, first, to determine the role of polymorphonuclear neutrophil (PMN) adherence to arterial and venous graft tissue after stimulation with thrombin or dilation procedure (mechanical distention or papaverine treatment); second, to investigate the mechanisms of PMN adherence after dilation; and third, to determine the role of enhanced leukocyte adherence to vascular injury.
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Material and Methods
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Preparation of Human Saphenous Vein and Mammary Artery Rings
Specimens of SV or IMA grafts were used from 38 patients at the time of coronary artery bypass grafting. This was done with the approval of our hospital's institutional review board (No. 1997/02). All patients signed informed consent. Specimens were transported immediately to the laboratory in cardioplegic solution (Bretschneider solution). Coronary arteries were placed into ice-cold Krebs–Henseleit solution consisting of the following (in mmol/L): NaCl, 118; KCl, 4.75; CaCl2
· 2 H2O, 2.54; KH2PO4, 1.19; MgSO4
· 7 H2O, 1.19; NaHCO3, 12.5; and glucose 10.0. Isolated vessels were cleaned of fat and connective tissue and cut into rings of 2 to 3 mm in length for determination of PMN–endothelial adherence and for subsequent studies in organ chambers.
For the SVs two different types of vessel treatments were analyzed: (1) isolated regular veins; (2) SVs that were treated with high-pressure distention. In detail, side branches of the venous grafts were occluded. Grafts were filled with Bretschneider solution (Custodiol; Köhler Chemie, Alsbach-Hähnlein, Germany, containing the following in mmol/L: NaCl, 15; KCl, 9; MgCl2, 4; histidine-HCl, 18; histidine, 180; tryptophan, 2; mannitol, 30; CaCl2, 0.015; and potassium hydrogen-2-oxopentandioate, 1), and high pressure of approximately 200 to 300 mm Hg was administered for up to 1 minute with a syringe. For IMAs three different vessel treatments were tested: (1) regular untreated IMA, (2) internal papaverine administration into the IMA, and (3) external papaverine treatment to the IMA.
Human Neutrophil Isolation
Blood was taken from the cubital vein before coronary artery bypass graft surgery. Human neutrophils were isolated by a modified method as described by Boyum [7]. Isolation procedure did not activate neutrophils (ie, L-selectin shedding could not be observed). Polymorphonuclear neutrophil preparations obtained by this method were greater than 95% pure and greater than 95% viable. The PMN pellet was finally suspended in 2 mL of phosphate-buffered saline solution, and the number of cells was counted.
Human Polymorphonuclear Neutrophil Adherence to Stimulated Venous and Arterial Vascular Endothelium
Polymorphonuclear neutrophils isolated by the procedure described above were labeled with a fluorescent dye (PKH2-GL; Sigma Immunochemical Co, Deisenhofen, Germany) according to the method of Yuan and Fleming [8] and described previously [9].
Human venous and arterial rings were prepared as described above. Rings were carefully opened with microsurgery scissors, and placed with the endothelial surface up in a culture dish filled with 3 mL of oxygenated Krebs–Henseleit buffer (37°C). To stimulate endothelial cells, graft rings were incubated with 0.5 to 2 U/mL thrombin for 10 minutes or vessels were used after dilation procedures (ie, Bretschneider solution dilation, papaverine treatment). After incubation, venous or arterial ring segments were removed and placed in another cell culture dish filled with fresh oxygenated Krebs–Henseleit solution. These rings were incubated with saline and, as a vehicle, fucoidin (Sigma Chemical Co, Deisenhofen, Germany) or monoclonal antibody directed against P-selectin (PDL; Protein Design Laboratories, Mountain View, CA) for 10 minutes. Labeled PMNs (400,000 PMNs/mL) were then gently added to the bottom of cell culture dishes and incubated for 20 minutes. During this period, culture dishes were agitated in a shaker bath (70 movements/min) at 37°C to apply shear force to the endothelial surface. After incubation, graft rings were removed, placed onto glass slides, and covered with a coverslip. Labeled PMNs, which adhered to the endothelial surface, were counted using epifluorescence microscopy (Zeiss, Göttingen, Germany). Adherent neutrophils of five regions of each vessel segment were randomly counted by two blinded investigators and expressed as mean number of PMNs per square millimeter of endothelial surface.
Scanning Electron Microscopy
Graft rings (ie, normal or after dilation procedure) were prepared as described above. After adhesion experiments, venous grafts were fixed overnight at 40°C in 2.5% glutaraldehyde in 0.13 mol/L cacodylate buffer (pH 7.4). After being rinsed twice in buffer, specimens were treated with 1% osmium tetroxide at 40°C for 1.5 hours for fixation, rinsed three times in cacodylate buffer, and dehydrated through graded concentrations of acetone. The specimens were critical point–dried from 100% ethanol with liquid CO2. After specimens were dried, they were divided to expose the luminal surface, mounted on stubs, and sputter-coated with gold. The samples were viewed and photographed at 10 kV with a scanning electron microscope (Hitachi Instruments, Krefeld, Deutschland). Photomicrographs were taken at x1,000 magnifications.
Isolated Human Vein Ring Study
Isolated human SV rings were mounted between two stainless-steel hooks, and suspended in organ chambers connected to force-displacement transducers (TSE-Instrument Co, Bad Homburg, Germany) to record changes in isometric force on an oscillographic recorder (TSE-Instrument Co). The chambers were filled with 18 mL of Krebs–Henseleit buffer, kept at 37°C, and oxygenated with 95% O2 and 5% CO2. Graft rings were progressively stretched to give a preload of 2.0 g of force and equilibrated for 90 minutes before starting the experimental protocol. During this period, the Krebs–Henseleit buffer in the organ chambers was replaced every 20 minutes. After equilibration, rings were contracted with 100 nmol/L U-46619 (9,11-epoxymethano-PGH2; Sigma Chemical Co), a thromboxane A2 mimetic, to generate a precontraction. Once a stable plateau was obtained, the calcium ionophore A23187 was added to the chambers in cumulative concentrations (ie, from 0.1 to 100 nmol/L). After stabilization, rings were washed and allowed to equilibrate for 15 minutes to reach baseline force. After determination of initial vascular response, PMN (106/mL) were added to the organ bath. Vasoconstriction was analyzed over the course of 30 minutes. Thereafter, the endothelium-independent vasodilator sodium nitroprusside in cumulative concentrations (ie, 0.1, 1, 10, and 100 µmol/L) was added. The maximum relaxation after PMN administration was calculated as the percent reduction of the U46619-induced tone.
Statistical Analyses
All values are presented as mean ± standard error of the mean, based on n independent experiments. All data were subjected to analysis of variance followed by the Fisher's exact test for evaluation of the difference between groups. Probabilities of 0.05 or less were considered to be statistically significant for difference between groups. All computations were carried out with StatView (Abacus Concept Inc, Berkeley, CA).
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Results
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Adherence of Unstimulated Polymorphonuclear Neutrophils to Thrombin-Stimulated Saphenous Vein Endothelium
To upregulate P-selectin on the endothelial surface, we stimulated the venous vascular endothelium with thrombin (from 0.5 to 2 U/mL) for 10 minutes. Only a few PMNs adhered to unstimulated endothelium. However, addition of PMNs to thrombin-stimulated venous graft endothelium resulted in a significant, dose-dependent increase of PMN adherence. Selectin-mediated interaction of leukocytes and vascular endothelium can be blocked by carbohydrates like sialyl Lewisx, Lewisx, or fucoidin. Therefore, addition of fucoidin (0.1 mg/mL) inhibited PMN adherence to thrombin-stimulated venous graft endothelium (Fig 1A). Similarly, monoclonal antibody directed against P-selectin reduced PMN adherence to thrombin-stimulated venous graft endothelium (data not shown). Further, increased PMN adherence (ie, threefold) could be observed on the IMA endothelial surface when stimulated with thrombin (from 0.5 to 2 U/mL) for 10 minutes. In contrast, only a few PMNs adhered to unstimulated endothelium in IMA. Addition of fucoidin (0.1 mg/mL) inhibited PMN adherence to thrombin-stimulated endothelium (Fig 1B). These results indicate that fucoidin effectively inhibits PMN adherence to arterial vascular endothelium activated by thrombin. However, the number of adhering PMN was lower when compared with PMN adherence on venous graft endothelium after thrombin stimulation.
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Fig 1. (A) Bar graph showing adherence of unstimulated human polymorphonuclear neutrophils either to unstimulated human saphenous vein graft endothelium (control) or to thrombin (0.5 to 2 U/mL) -stimulated vascular graft endothelium. Fucoidin, as a selectin-blocking carbohydrate, significantly attenuated polymorphonuclear neutrophil adherence to thrombin-stimulated saphenous vein endothelium. (B) Bar graph showing adherence of unstimulated human polymorphonuclear neutrophils either to unstimulated human mammary artery graft endothelium (control) or to thrombin (0.5 to 2 U/mL) -stimulated arterial endothelium. An antiselectin carbohydrate (fucoidin) significantly attenuated polymorphonuclear neutrophil adherence to thrombin-stimulated human mammary artery graft endothelium. Values are mean ± standard error of the mean. Twenty-four to 29 saphenous vein graft segments of 12 patients were studied. Twenty-six to 30 human mammary artery graft segments of 14 patients were studied (*p < 0.05; **p < 0.01; ***p < 0.001).
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Adherence of Unstimulated Polymorphonuclear Neutrophils to Saphenous Vein Endothelium After Dilation Procedure
Because vascular endothelium might be activated after dilation with Bretschneider solution to gain better flow in the coronary graft, we further quantified PMN adherence to mechanically dilated venous graft endothelium. Figure 2
summarizes the results of unstimulated PMN adherence to dilation procedure–stimulated venous graft endothelium. Polymorphonuclear neutrophil adherence was significantly enhanced to the endothelium after the dilation procedure. Figure 3
demonstrates the photomicrograph of fluorescence-labeled PMNs adhering to normal or dilated venous graft endothelium. The increased adhesiveness of unstimulated PMNs to stimulated graft endothelium was significantly attenuated by treatment with fucoidin. Addition of fucoidin (0.01, 0.1, or 0.5 mg/mL) or similar monoclonal antibody directed against P-selectin (10 µg/mL) inhibited PMN adherence to the dilated endothelium in a dose-dependent manner (Fig 2). At a dosage of 0.1 mg/mL, a maximal inhibitory effect was obtained (ie, about 60% inhibition). Increasing the concentration of fucoidin greater than 0.1 mg/mL did not further inhibit PMN adherence.
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Fig 2. Bar graph showing adherence of unstimulated human polymorphonuclear neutrophils either to unstimulated saphenous vein endothelium (control) or to Bretschneider solution–dilated saphenous vein endothelium. An antiselectin carbohydrate (fucoidin) significantly attenuated polymorphonuclear neutrophil adherence to dilation-stimulated saphenous vein endothelium. Values are mean ± standard error of the mean. Twenty-five to 34 saphenous vein graft segments of 14 patients were studied (*p < 0.05; **p < 0.01; ***p < 0.001).
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Fig 3. Photomicrograph showing fluorescence microscopy analysis of unstimulated human polymorphonuclear neutrophils (PMNs) adhering to either unstimulated saphenous vein endothelium (control; A) or to Bretschneider solution–dilated saphenous vein endothelium (B). Only a few fluorescence-labeled neutrophils are adhering to normal venous graft endothelium. In contrast, increased neutrophil adherence is detectable in dilated saphenous vein graft. Arrows indicate adhering neutrophils.
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Scanning Electron Microscopy Analysis of Polymorphonuclear Neutrophil Adherence to Saphenous Vein Endothelium After Dilation Procedure
Scanning electron photomicrographs of unstimulated human PMNs adhering to either unstimulated SV endothelium (control) or to Bretschneider solution–dilated SV endothelium are presented in Figure 4. In control grafts well-preserved endothelium was visible. Endothelial cell nuclei were visibly protruding into the lumen. Interendothelial cell junctions are intact. In contrast, endothelial disruption is detectable, as well as increased PMN adherence, in dilated SV. Endothelial injury, contracted endothelial cells, and loss of endothelium were visible, as presented in Figure 4.
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Fig 4. Scanning electron micrograph of unstimulated human polymorphonuclear neutrophils (PMNs) adhering to either unstimulated saphenous vein graft endothelium (control; A) or to Bretschneider solution–dilated saphenous vein graft endothelium (B). In control graft, well-preserved endothelium is visible. Endothelial cell nuclei are visible protruding into the lumen. Interendothelial cell junctions are intact. In contrast, endothelial disruption is detectable, as well as increased neutrophil adherence in dilated saphenous vein graft. Endothelium has been lost, exposing the subendothelial connective tissue. Arrows indicate adhering neutrophils.
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Adherence of Polymorphonuclear Neutrophils on Internal Mammary Artery Endothelium After Papaverine Treatment
Papaverine treatment is routinely performed to prevent vasospasm of mammary arteries in coronary artery bypass graft surgery. Administration of papaverine to IMA can be performed externally or internally. Because vascular endothelium might be activated after papaverine treatment, we further quantified PMN adherence to papaverine-stimulated human IMA endothelium. Figure 5
summarizes the results of unstimulated PMN adherence to papaverine-stimulated arterial endothelium. External papaverine treatment did not affect PMN adherence to vascular endothelium. In contrast, PMN adherence was significantly enhanced after internal papaverine treatment. The increased adhesiveness of unstimulated PMNs to stimulated arterial graft endothelium was significantly attenuated by treatment with fucoidin or monoclonal antibody directed against P-selectin (data not shown). Thus, selectin-blocking carbohydrate or monoclonal antibody markedly attenuates PMN adherence to mammary artery endothelium after internal papaverine administration.
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Fig 5. Bar graph showing adherence of unstimulated human polymorphonuclear neutrophils either to unstimulated human mammary artery graft endothelium (control) or to papaverine-dilated mammary artery graft endothelium. An antiselectin carbohydrate (fucoidin) significantly attenuated polymorphonuclear neutrophil adherence to papaverine-stimulated human mammary artery graft endothelium. Values are mean ± standard error of the mean. Eighteen to 22 human mammary artery graft endothelium segments of 9 patients were studied (**p < 0.01).
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Endothelial Function After Dilation Procedure of Saphenous Vein
To evaluate whether endothelial function is altered after dilation procedure, we tested the ability of precontracted venous rings to relax in response to A23187 or sodium nitroprusside treatment in normal or dilated venous grafts after mechanical force treatment. Normal venous rings elicited normal relaxation to both A23187 and sodium nitroprusside, whereas after dilation of SV with Bretschneider solution, endothelium-dependent relaxation to A23187 was markedly attenuated; however, endothelium-independent relaxation in response to sodium nitroprusside treatment was not altered (Fig 6A). Thus, endothelium-dependent relaxation to A23187 was significantly reduced after the dilation procedure. Similar results were obtained with mammary artery rings when internal papaverine treatment was performed. However, when PMN were added to dilated graft rings, we observed significant vascular contraction within 10 to 15 minutes (Fig 6B). Further, when the vascular response to A23187 was tested after PMN incubation, we could detect almost no vasorelaxation, indicating severe endothelial dysfunction.
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Fig 6. Relaxation of unstimulated saphenous vein rings (normal) or Bretschneider solution–dilated saphenous vein rings in vitro to A23187 after precontraction with U46619 (A). Endothelial dependent-relaxation after A23187 was significantly reduced after dilation procedure when compared with normal venous grafts. In contrast, endothelial-independent relaxation after sodium nitroprusside did not demonstrate any significant differences when normal venous grafts were compared with grafts after high-pressure distention (B). Clearly, dilated saphenous vein rings demonstrated significant endothelial dysfunction when compared with normal venous graft rings, indicating loss of nitric oxide release after dilation procedure. Values are mean ± standard error of the mean. Twenty-eight to 34 saphenous vein graft segments of 15 patients were studied (*p < 0.05).
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Comment
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In the present study we demonstrated increased PMN adherence after thrombin activation to the vascular endothelium of human SV grafts or IMA grafts. Furthermore, increased PMN–endothelium interaction could be observed in vitro when SV were mechanically dilated with Bretschneider solution. Increased PMN adherence could be blocked by fucoidin, a selectin-blocking carbohydrate, or by P-selectin monoclonal antibody, indicating the involvement of selectins or their ligands. The increase in PMN adhesion was accompanied by endothelial dysfunction of the dilated venous grafts. Treatment of IMA with papaverine is usually performed to prevent spasm after aortocoronary bypass graft surgery. We showed that this procedure leads to a marked increase of PMN adherence when papaverine was administered directly into the vessel lumen. However, external treatment with papaverine did not increase PMN adherence. As shown for SV, PMN adherence could be blocked either by P-selectin monoclonal antibody or fucoidin also in IMA, indicating the involvement of selectins or their ligands.
Expression of adhesion molecules is low in SVs that have been harvested at the time of operation before coronary artery bypass grafting [10, 11]; however, they can be upregulated by mechanical and chemical stimuli [12]. In accordance with the results of our study, Chello and colleagues [13] have shown that high distention pressure stimulates the expression of endothelial adhesion molecules in SV and subsequently adhesion of PMNs to the vessel wall. High distention pressure causes loss of endothelium [3]. However, in our study with moderate-pressure distention we observed reduction of the vascular endothelium for up to 10% to 15%. Excessive tension in the vein wall causes separation of endothelial cells, disrupts the medial smooth muscle cells, and stretches the intima so that the endothelial cells are separated and contracted [14]. After perfusion of the graft, some of these endothelial cells are easily washed away, which might increase vasoconstriction. If the endothelium is lost during harvesting, the denuded areas will become covered by regenerated endothelium after 1 to 2 weeks [15]. However, this neoendothelium is laid over a carpet of platelets and fibrin that has been deposited on the thrombogenic basement membrane. Later, this thrombus is replaced by proliferating smooth muscle cells. Platelets are known to release various factors that are implicated in the development of intimal hyperplasia, which, if progressive, may lead to graft occlusion. A variety of growth factors are involved in endothelial regeneration and subsequent intimal hyperplasia [16]. Thus, preservation of the endothelial surface is vital to graft integrity and long-term function.
Inflammatory responses after vascular injury or myocardial ischemia–reperfusion lead to a sequence of events involving PMN adherence to the vascular endothelium followed by PMN activation, emigration, and phagocytosis [17]. During this process, activated PMNs that adhere to the vascular wall evoke a significant endothelial cell injury characterized by diminished endothelium-dependent vasorelaxant responsiveness [18]. In our studies we were able to demonstrate reduced endothelial-dependent relaxation to A23187 after mechanical distention, indicating endothelial dysfunction and further, vascular injury or endothelial dysfunction after PMN adherence. The importance of endothelial (dys)function in patency of bypass grafts has been shown. Polymorphonuclear neutrophil adhesion to the endothelium of IMA is decreased in comparison to SV, and this is a result of enhanced release of nitric oxide at this level [19]. Nitric oxide modulates the vascular tone and the leukocyte–endothelial cell interaction by suppressing upregulation of adhesion molecules on endothelial cells and PMNs [20]. Thus, nitric oxide (or expression of nitric oxide synthase) may be significantly involved in regulation of adhesion molecule expression on endothelium from SV or IMA. Furthermore, endothelial nitric oxide synthase gene transfer inhibits in vitro bypass graft disease in intact human SV tissue, presenting a possibly therapeutic target for treatment of bypass graft disease [21].
In our studies we were able to further demonstrate reduced endothelial-dependent relaxation to treatment by A23187 after mechanical distention, which indicates endothelial dysfunction. Activated PMNs can also elicit an endothelium-dependent vascular contraction by means of enhanced degradation of basal nitric oxide in response to superoxide free radicals released from PMNs [22]. It has been demonstrated that PMN-induced vascular contraction was alleviated by a specific antibody directed against a ß2-integrin adhesion molecule (CD18) [23].
Polymorphonuclear neutrophil recruitment to inflammatory tissue is mediated by the sequential action of multiple adhesion molecules [24–26]. Initial interaction between PMNs and the vascular endothelium (ie, PMN rolling) is mediated by the selectin adhesion molecule family [24]. When endothelial cells are stimulated with inflammatory agents such as thrombin, histamine, or hydrogen peroxide, P-selectin is rapidly translocated to the endothelial surface from Weibel-Palade bodies (ie, within 5 to 10 minutes) [27]. P-selectin has been shown to be translocated to the endothelial surface 10 minutes after reperfusion of ischemic coronary arteries [26, 28]. Furthermore, P-selectin upregulation occurred on the endothelium of large epicardial coronary arteries as well as postcapillary venules [29]. In the present study, SV or IMA, which were stimulated with thrombin or dilation procedure, exhibited enhanced PMN adherence to the vascular endothelium. Increased adhesion was significantly attenuated by P-selectin monoclonal antibody or selectin-blocking carbohydrate. L-selectin is constitutively expressed on the surface of leukocytes and is also known to play an important role in the initial rolling step of PMN during inflammation [23, 30, 31]. In contrast, E-selectin is expressed through de novo synthesis for 4 to 6 hours after activation of endothelium by cytokines (eg, tumor necrosis factor 
, interleukin 1ß), or by endotoxin, or ischemia–reperfusion [32]. In this regard, E-selectin may not be as critical as other selectins in our experimental setting as we performed the adhesion experiments within 2 hours after graft isolation.
Some studies have indicated that selectin adhesion molecules have common carbohydrate ligands (eg, sialyl Lewisx, sialyl Lewisa, PSGL-1, or others) [24]. It has been shown that a soluble sialyl Lewisx-containing oligosaccharide (SLex-OS), but not its nonsialylated analog Lex-OS, significantly protected against endothelial dysfunction, myocardial necrosis, and PMN adherence to coronary endothelium subjected to ischemia–reperfusion [9]. In the present study, fucoidin or P-selectin monoclonal antibody significantly inhibited PMN adherence to thrombin-stimulated, papaverine-treated, or dilated vascular graft endothelium. These results provide clear evidence that initial PMN–endothelial interaction between selectins and their ligands plays an important role in PMN-induced vascular injury.
In conclusion, we have demonstrated increased adherence of PMNs to the endothelial surface in isolated human SV and IMA grafts stimulated with thrombin or dilation procedure used for coronary artery bypass graft surgery. Polymorphonuclear neutrophil adherence was significantly attenuated by selectin-blocking agents (ie, monoclonal antibody or fucoidin), indicating that selectin-mediated PMN–endothelial interaction plays an important role in damage of venous or arterial grafts used in coronary artery bypass graft surgery. Our results demonstrate increased PMN–endothelium interaction in distended venous or arterial grafts, which might indicate that blocking of PMN adhesion could improve short-term and long-term results of SV or IMA grafts.
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Acknowledgments
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This study was partly supported by grant Bu 819/3–1 of Deutsche Forschungsgemeinschaft. The authors gratefully acknowledge Stefanie Becker and Till Diergarten for their excellent technical assistance during the course of the experiments.
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Footnotes
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* Both authors contributed equally to the submitted work. 
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Abstract
Introduction
