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Original Contribution
Release and Elimination of Soluble Vasoactive Factors during Percutaneous Coronary Intervention of Saphenous Vein Grafts: Analys
November 2005
The no-reflow phenomenon is defined as a severe reduction in antegrade coronary flow in the absence of epicardial vessel obstruction.1 The presence of no-reflow substantially increases the risk of major adverse clinical events (MACE) in percutaneous coronary artery interventions (PCI), particularly in saphenous vein grafts (SVG).2,3 Mechanisms underlying no-reflow are not completely understood. Several hypotheses have been proposed, including microvascular spasm, in situ thrombosis and distal embolization. Distal protection devices are designed to prevent no-reflow by preventing the distal embolization of materials released during SVG PCI. Several clinical trials have shown that distal protection devices substantially reduced MACE in SVG PCI.4–6 Webb et al. reported a 3.9% MACE rate in patients in whom the PercuSurge Guardwire®(PercuSurge, Minneapolis, Minnesota) distal protection device was used as compared to a historical 17.3% MACE rate in SVG PCI.I6 The SAFER Trial randomized patients to the GuardWire device versus conventional angiogplasty wire (no distal protection devices). There was a 42% relative MACE rate reduction in patients who received the GuardWire distal protection device.4 Distal protection devices may be filter devices5 that trap debris or distal occlusion and aspiration systems, such as the GuardWire. The GuardWire is a device for transient distal vessel occlusion during balloon dilatation or stent placement that allows recovery of any liberated plaque material by aspiration of SVG contents before the restoration of flow. The GuardWire system has demonstrated its ability to recover cholesterol crystals, foam cells and other plaque constituents.6 One potential advantage of distal occlusion devices is that they eliminate both debris and soluble factors that can lead to no-reflow. Recent studies have indicated that soluble vasoactive compounds may contribute to the no-reflow phenomenon.7,8 Vasodilators such as calcium channel blockers, adenosine and nitroprusside reverse no-reflow in 50–80% of cases, suggesting that vasospasm plays a significant role in no-reflow.7,8 In addition, thrombotic factors released during PCI potentially increase in situ thrombosis that leads to no-reflow. Recent studies9–12 demonstrated an association between tissue factor and no-reflow in acute coronary syndrome (ACS). The release of vasoactive soluble factors and the ability of the GuardWire to recover soluble elements that could be released during SVG interventions have not been investigated before and are the focus of the present study.
Methods
Patients and procedures. Twenty-eight consecutive patients underwent catheter-based interventions on 34 SVG lesions using the GuardWire. The procedural details of the use of the GuardWire distal protection device have been previously illustrated. Systemic anticoagulation for the procedure was obtained with unfractionated heparin. Glycoprotein (GP) IIb/IIIa receptor antagonists were used at the discretion of the operators. The procedure was conducted using the GuardWire in the usual manner.4 After crossing the lesion with the 0.014 inch hollow core GuardWire, a baseline coronary artery blood sample was drawn using the PercuSurge Export Catheter (Medtronic, Inc., Minneapolis, Minnesota) prior to inflation of the GuardWire. The distal occlusion balloon was then inflated and complete occlusion of the SVG was documented by demonstrating absence of flow in the SVG. Predilatation with an angioplasty balloon or stent deployment was then performed. After withdrawal of the catheter, the Export Catheter was advanced over the GuardWire until the catheter tip was just proximal to the occlusive balloon. The Export Catheter was connected to an evacuated 20 ml syringe and used to aspirate the contents of the SVG. Two 20 ml blood samples were obtained. Each sample was placed on ice and processed immediately. Serum and plasma were obtained and stored in -80°C until analysis. If there was a need for a second stent, the same procedure was followed and 2 other aspiration samples were obtained. At the conclusion of the procedure, patients were admitted for overnight stay. Standard post-stent therapy (aspirin 325 mg daily, clopidogrel 300 mg load, followed by 75 mg daily for a minimum of 4 weeks) was commenced. Serum levels of creatine phosphokinase and its myocardial fraction (CK and CK-MB) were drawn routinely 8 hours after the procedure. A 12-lead electrocardiogram was routinely recorded immediately and the morning after the procedure. Clinical follow-up was conducted by the referring physicians and the information was gathered prospectively from the patients’ medical records. If access to medical records could not be obtained, patients were contacted by the investigators via telephone. Clinical parameters included death, myocardial infarction, emergent or need for target vessel revascularization.
Analysis of vasoactive factors. Blood samples were collected on ice and centrifuged immediately. Serum or plasma was stored at -80°C until the time of assay. Blood samples for measurement of plasminogen activator inhibitor-1 (PAI-1), thrombin/antithrombin III complex (TAT), prothrombin fragment F1+2 (F1+2), tissue factor (TF), soluble CD40 ligand (sCD40L), and serotonin were collected in tubes containing 0.105 mol/L acidified sodium citrate. Soluble E-selectin and Endothelin-1 (ET-1) were measured in serum. PAI-1 antigen levels were determined by using a 2-site ELISA (Biopool AB, Umea, Sweden) as previously described.13 Thrombin/antithrombin III complex (Dade Behring, Newark, Delaware), prothrombin fragment F1+2 (Dade Behring), tissue factor (American Diagnostica, Stamford, Connecticut), soluble CD40 ligand (R and D, Minneapolis, Minnesota), soluble E-selectin (R and D), Endothelin-1 (R and D), and serotonin (ALPCO, Windham, New Hampshire) levels were determined using commercial ELISA kits.
Satistical analysis. Death was defined as the occurrence of death from any cause. Myocardial infarction was defined as the occurrence of an elevated CK-MB fraction > 3 times the upper limit of normal, or the development of pathological Q-waves on the ECG. Technical success was defined as delivery of the GuardWire system to the target site, followed by successful balloon angioplasty or stent placement, aspiration and deflation of the distal occlusive balloon. Other clinical criteria included postprocedure TIMI flow grades and the occurrence of postprocedural stroke. Levels of soluble vasoactive factors were expressed as means ± standard deviation. Results were compared at baseline, first aspirate versus second aspirate using the Student’s t-test. A p-value (Integrilin®, COR Therapeutics, Inc., South San Francisco, California) was used in 13 of these cases, while tirofiban (Aggrastat®, Merck & Co., Inc.,West Point, Pennsylvania) was infused in the remaining 3 cases. In all of these cases, patients had been started on the GP IIb/IIIa receptor antagonist upon presenting to the hospital, prior to their transfer to the catheterization laboratory. In 10 patients, additional adjunctive therapy included sodium nitroprusside; a dose of 100 mcg was administered intracoronary through the guiding catheter before stent deployment and a similar dose was again administered after GuardWire balloon deflation. The mean GuardWire occlusion time was 6 minutes and 22 seconds. Mean hospital length of stay was 3 ± 3 days. Mean duration of follow-up was 6 ± 3 months. Over this period, MACE occurred in 5 patients (18%). They included 1 death, 3 myocardial infarctions and 1 target lesion revascularization. The death was due to acute stent thrombosis that occurred 30 hours after initial stent deployment. The 3 myocardial infarctions were diagnosed by myocardial enzyme assay. No new pathologic Q-waves were seen on the postprocedural electrocardiogram. One patient presented with angina symptoms 3 months after the initial procedure. Diagnostic cardiac catheterization revealed significant in-stent restenosis at the site of the previous stent deployment. Cutting balloon angioplasty was performed and yielded good results.
The analysis of blood samples obtained during SVG interventions are the focus of this study. Three samples were obtained from each patient. The first sample was obtained before stenting by aspirating 20 ml from the Export Catheter after systemic anticoagulation with unfractionated heparin. After stent deployment, the Export Catheter was advanced over the wire and used to aspirate two 20 ml blood samples. These 3 samples were then sent for quantitative ELISA assays of several soluble vasoactive substances. Three groups of soluble factors were analyzed. They are: (1) Vasoconstrictive factors: endothelin (ET) and serotonin (5-HT); (2) thrombotic factors: tissue factor (TF), plasminogen activator inhibitor (PAI-1), thrombin/antithrombin III complex (TAT), and prothrombin fragment F1+2 (F1+2); and (3) inflammatory factors: soluble CD40 ligand (sCD40L) and soluble E-selectin.
The levels of these soluble factors showed a significant increase in the first Export Catheter aspirate as compared to baseline. The second aspirate showed decreasing levels when compared to the first aspirate. For vasoconstrictive factors, ET levels were 3 times higher in the first aspirate compared to baseline (6.7 ± 2.1 pg/ml versus 1.6 ± 0.9 pg/ml; p p = 0.031), then decreased to 60.9 ± 45.6 ng/ml. For thrombotic factors, TF levels were 4.5 times higher in the first aspirate (143.8 ± 93.5 ng/ml versus 26.5 ± 24.1 ng/ml; p = 0.005), then decreased to 35.3 ± 22.5 ng/ml in the second aspirate. PAI-1 levels, however, did not show any significant change in the 3 samples (baseline 8.1 ± 2.3 ng/ml, first aspirate 10.4 ± 2.9 ng/ml, and second aspirate 9.9 ± 3.1 ng/ml. P-values were 0.19 and 0.27, respectively). TAT levels were 2.4 times higher in the first aspirate (13.1 ± 2.9 mg/L versus 5.4 ± 0.5; p p = 0.08) in the second aspiration. F1+2 levels were 2.4 times higher in the first aspirate (2.1 ± 0.5 nmol/L versus 0.9 ± 0.3; p p = 0.57). A substantial amount of inflammatory factors were also released by stenting in the SVGs. sCD40L levels were 2,181 ± 1,688 pg/ml at baseline and increased to 4,866 ± 3,185 in the first aspirate (123% increase, p = 0.003). sCD40L levels remained elevated in the second aspiration (4,449 ± 2,439; p = 0.009 as compared to baseline). Soluble E-selectin levels increased modestly after stenting from 55.9 ± 5.8 pg/ml at baseline to 69.7 ± 6.2 (25% increase; p p = 0.06 as compared to baseline). Table 3 and Figure 1 illustrate these results.
There were no significant differences in the levels of these factors between patients with or without GP IIb/IIIa inhibitors. However, the number of patients in this study was not sufficient to conduct any meaningful statistical analysis.
Discussion
It has been hypothesized that coronary intervention results in a significant release of vasoactive factors that may be responsible for many adverse events.10,11 To our knowledge, there are no data available to confirm this hypothesis in human subjects. Occlusive distal protection devices such as the GuardWire have provided a unique opportunity to study the effects of stenting on the release of vasoactive factors in situ in human subjects. Our observation demonstrates for the first time that stenting in SVGs results in a significant elevation of many vasoconstrictive, thrombotic, and inflammatory factors. These factors can potentially mediate vasospasm, in situ thrombosis, and in situ inflammatory response, which may precipitate various post-PCI complications.
The measurement of these substances offers additional insight into the humoral milieu that evolves during SVG interventions. ET and 5-HT are known to be released by activated platelets, endothelial cells, macrophages and smooth muscle cells. By their vasoconstrictive effects on the microvasculature, ET and 5-HT may be responsible, at least in part, for procedural adverse events such as the no-reflow phenomenon. Reduction in platelet activation may lead to a reduction in the release of these substances, hence a reduction in complication rates. Antiplatelet agents, namely GP IIb/IIIa receptor antagonists, lead to improved procedural outcomes in native coronary interventions. The total number of patients in this study is not sufficient to study whether these platelet inhibitors effectively lead to a reduction in vasoactive substance release. Analyses of data from multiple randomized trials, however, showed that GP IIb/IIIa receptor antagonists do not offer an added benefit in SVG interventions. It is possible that platelet activation during SVG intervention is so intense that their inhibition with GP IIb/IIIa receptor antagonists may not be enough to prevent significant vasoactive substance release, as shown by our study. An alternative explanation is that the majority of factors that are released originate from the significant amount of atherosclerotic plaque burden that is usually seen in SVGs. Hence, GP IIb/IIIa receptor antagonists may have little protective effect when used in SVG PCI.
Tissue factor is a membrane-bound glycoprotein that is not normally present in the circulation. Plasma levels of TF are substantially elevated in patients with ACS.9–11 Reduction in TF levels are seen after successful treatment of ACS. Interestingly, persistent elevation of TF in patients with treated ACS signifies higher subsequent MACE rates. A recent study by Bonderman and colleagues demonstrated that TF induces no-reflow in a porcine model.12 Balloon angioplasty and stenting result in plaque fracturing that may expose and release TF into the circulation. Previous studies have documented the elevation of TF levels in coronary sinus blood following PCI.9–11 The elevation of TF levels was accompanied by increased levels of thrombin/antithrombin complex. In the current study, we confirm the previous observation and provide further evidence that a significant amount of TF is released in situ immediately after PCI in SVGs. Elevation of TF was associated with increased levels of thrombin/antithrombin III complex and prothrombin fragment F1+2. These data provide compelling evidence to support the hypothesis that coronary intervention results in in situ thrombosis mediated by the release of TF and other thrombotic factors.
Inflammatory activity is directly associated with pathogenesis of atherosclerosis, thrombosis in ACS, and in-stent restenosis.14,15 Soluble CD40 ligand is a pro-inflammatory substance and is known to be released from activated platelets.16,17 In addition, it promotes blood coagulation by inducing the expression of tissue factor on monocytes and endothelial cells.18,19 Analysis from the CAPTURE Trial has demonstrated that elevation of soluble CD40 ligand levels indicates an increased risk of a cardiovascular event.20 Results from the current study are consistent with previous observations and directly demonstrate significant elevation of soluble CD40 ligand in situ immediately after PCI in SVGs. The data provide a link between PCI-mediated vascular injury/platelet activation and elevation of inflammatory factors. Elevation of these factors may contribute to various adverse cardiac events, including no reflow, myocardial infarction and restenosis after PCI.
The increase over baseline in vasoactive compounds in the first aspirate and the subsequent decrease shows that these substances are effectively cleared by the GuardWire distal protection system. To our knowledge, this is the first instance where this has been demonstrated experimentally in humans. It is well known that SVG interventions are accompanied by a higher incidence of complications when compared to interventions on native coronary vessels.3 The no-reflow phenomenon has a particularly higher incidence in SVG interventions.3 At first, attention was primarily given to embolization of particulate debris as a cause of the problem.6 Newer reports, however, point to the importance of soluble factors, namely coagulation and vasoconstrictive factors3,7,9,12,21 in the development of the no-reflow phenomenon. The effectiveness of the GuardWire system in eliminating vasoactive substances has been demonstrated by our study. Instances whereby such vasoactive substances are released in large amounts may be those where the GuardWire system can be especially beneficial. Studies to analyze vasoactive factors after deflation of the PercuSurge GuardWire are needed to further determine the effectiveness of the device.
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