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Ann Thorac Surg 2007;83:1374-1380
© 2007 The Society of Thoracic Surgeons
a Department of Cardiovascular Sciences, Units of Cardiac Surgery and Cardiology, University Campus Bio-Medico, Rome, Italy
b Interdisciplinary Center for Biomedical Research (CIR), University Campus Bio-Medico, Rome, Italy
Accepted for publication October 24, 2006.
* Address correspondence to Dr Chello, Department of Cardiovascular Sciences, Campus Bio-Medico University of Rome, Via Longoni 83, 00155 Rome, Italy (Email: m.chello{at}unicampus.it).
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Abstract |
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Methods: Thirty patients undergoing coronary surgery with cardiopulmonary bypass were randomized to treatment with either simvastatin (40 mg/day) or placebo for 3 weeks before surgery. A group of 15 patients undergoing off-pump coronary surgery served as controls. Blood samples for detection of serum cytokine levels were obtained before anesthesia, at the end of surgery, and at 4, 24, 48, and 72 hours postoperatively. Apoptosis and indices of activation were assessed in cultured neutrophils.
Results: Simvastatin significantly reduced the postoperative peak values of interleukin (IL)-6 and IL-8. The neutrophil apoptotic rate was significantly higher among neutrophils obtained from patients pretreated with simvastatin (p < 0.05 at both 8 and 24 hours) compared with placebo. Neutrophils from the statin group had depressed functional activity, as underscored by significantly lower values of CD11b (p < 0.01 at 24 hours) and a significantly less percentage of cells positive for nitro-blue tetrazolium (p < 0.01 at 12 and 24 hours) compared with placebo.
Conclusions: This randomized, double-blind study indicates that statin therapy before cardiopulmonary bypass is associated with reduction of circulating markers of inflammation and increased neutrophil apoptosis. These data support a routine inclusion of statins as an adjuvant pharmacologic therapy before cardiopulmonary bypass surgery.
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Introduction |
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Apoptosis is a process of regulated cellular death [3] that is mediated by a family of intracellular cysteine proteases or caspases [4] and represents the predominant process responsible for the resolution of the neutrophil-mediated inflammatory response. Neutrophils have the shortest half-life among leukocytes and normally survive for less than a day in the circulation [5] before undergoing morphologic and functional changes characteristic of apoptosis.
During culture, 50% to 70% of neutrophils undergo constitutive apoptosis by 20 hours [6]. As neutrophils undergo apoptosis, they lose surface adhesion molecules and the ability to secrete granular contents [7], and are subsequently ingested rapidly by macrophages and removed from an area of inflammation with minimal damage to the surrounding tissue. A delayed neutrophil apoptosis could therefore potentially prolong the systemic inflammatory response consequent to CPB [8].
Several studies have shown that 3-hydroxy-3 methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) induce apoptosis in a variety of cell lines [9–11], although their efficacy on inflammatory cells is still uncertain [12]. Because we have recently demonstrated that the functional lifespan of neutrophils is significantly prolonged after CPB as a consequence of a deferred apoptosis [13], in the present study we prospectively evaluated whether preoperative statin therapy with simvastatin is associated with effective stimulation of neutrophil apoptosis after coronary bypass grafting (CABG) surgery.
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Material and Methods |
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None of the study patients had taken any other cholesterol-lowering drugs for at least 1 year. The study protocol was approved by the local ethics committee. Informed consent was obtained from each patient.
Operative Procedure
All patients in the CPB groups and in the OP control group underwent CABG surgery performed using standard procedures. Induction and maintenance of anesthesia were similar for all patients and consisted of weight-related doses of fentanyl, midazolam, and pancuronium bromide. All operations were done through exposure of the heart with a median sternotomy incision.
CPB was performed in a standard fashion with the use of a hollow fiber oxygenator and a roller pump, with ascending aortic cannulation added to right atrium cannulation. During CPB, the hematocrit was maintained between 20% and 25%, and pump flows were kept between 2.0 and 2.5 L/(min · m2) to maintain a mean arterial pressure of 50 to 70 mm Hg. All patients were cooled to moderate hypothermia (mean, 32°C), and cardioplegic arrest was achieved with cold blood cardioplegia (4°C) infused into the ascending aorta.
Heparin was given at a dose of 300 IU/kg, obtaining an activated clotting time exceeding 400 seconds. Upon completion of the anastomoses, heparin effects were reversed by intravenous protamine sulfate (1 mg/300 IU of heparin) to achieve an activated clotting time similar to preoperative values.
Postoperative nonhemic volume expanders were routinely used. A standardized protocol for early postoperative care was followed in the intensive care unit. The perioperative need for blood products was determined on an individual, patient-by-patient basis. In general, blood transfusions were given in presence of a hemoglobin level of less than 9 g/dL, unless the patient was clinically considered at risk for decreased oxygen delivery. Inotropic agents were perioperatively used in patients with a cardiac index of less than 2.2 L/(min · m2), the target for cardiac index being 2.5 to 3.0 L/(min · m2).
Blood Sample Collection, Stocking, and Analysis
Blood samples for measurement of cytokine serum levels were collected through a radial arterial catheter before anesthesia, at the end of surgery, and at 4, 24, 48, and 72 hours postoperatively. All blood samples were drawn in prechilled vacuum tubes containing ethylenediaminetetraacetic acid for determination of interleukin-8 (IL-8), IL-6, and tumor necrosis factor-
(TNF-). The samples were immediately centrifuged at 4°C and stored at –80°C until assayed. Measurements of IL-8, IL-6, and TNF- were by means of commercially available enzyme-linked immunosorbent assays according to the supplier’s recommendations (R&D Systems, Abingdon, UK). All samples were measured in duplicate. The effect of hemodilution during CPB was corrected as follows: corrected concentration = measured concentration during CPB x (hematocrit before the operation/hematocrit during CPB). Neutrophil apoptosis was evaluated on neutrophils from samples drawn 18-hour postoperatively.
Neutrophil Isolation
Neutrophils were isolated by Ficoll-Hypaque density gradient centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes [8]. The final cell preparation had 98% ± 2% neutrophils. The neutrophils were maintained on ice in Hanks’ balanced salt solution at 1 to 5 x 106 cells/mL until used. Isolated neutrophils were more than 99% pure, as assessed by Wright’s stained cytocentrifuge preparation, and more than 99% viable, as assessed by exclusion of trypan blue.
Assays of Neutrophil Activation
Neutrophil CD11b expression
Neutrophil CD11b expression was detected by indirect immunofluorescence and flow cytometry, as previously described [8]. In the flow cytometry studies, the logarithmic mean fluorescence values obtained from the histograms were converted mathematically into a relative fluorescence value and expressed as a percentage increase from the observed baseline values.
Nitro-blue tetrazolium
One specific form of neutrophil activation is the capacity to reduce nitro-blue tetrazolium (NBT) dye, which is associated with an enhanced generation of superoxide anion radicals. The NBT test was performed on fresh blood as previously described [15]. The percentage of neutrophils containing formazan deposits of at least the size of a lobe of the nucleus was designed as positive NBT score.
Assessment of Apoptosis
Morphologic criteria
Neutrophil cell morphology was analyzed in duplicate independently by two blinded investigators under oil immersion light microscopy. The polymorphonuclear (PMN) cells were considered apoptotic if they showed dense condensation of chromatin in the form of either a single nucleus or nuclear fragments not connected by strands. At least 500 cells were counted per slide, and the results were expressed as the percentage of PMN cells on each slide that met the criteria for apoptosis.
Annexin V-fluorescein isothiocyanate binding
PMN apoptosis was also assayed by fluorescein isothiocyanate-(FITC)-labeled recombinant human annexin V that binds to phosphatidylserine exposed on the surface of apoptotic cells. The stock annexin V was diluted 1:200 with binding buffer and then 25 µL was added to 75 µL of the recovered cell samples. After a 5-minute incubation at room temperature, these samples were fixed by the addition of 100 µL of 3% formaldehyde in phosphate-buffered saline before analysis using a dual filter fluorescence microscope.
Western Blot Analysis
Western blot analyses using commercially available primary antibodies (Cell Signaling, New England Biolabs, Ipswich, MA) were performed with cellular lysate after 18 hours of incubation to examine the Fas, Fas ligand (Fas-L), caspase 3 (CPP32), and interleukin-1ß–converting enzyme (ICE) proteins [13]. Bands were detected by chemiluminescence, and optical density readings were obtained by Quantity One software (Bio-Rad, Hercules, CA) for IBM Corp (Somers, NY).
Assessment of caspase activity
Caspase activity was measured in vitro by the cleavage of the fluorogenic substrate Asp-Glu-Val-Asp (DEVD)-7-amino-4-methyl-coumarin (DEVD-AMC) in a continuous fluorometric assays as described previously [13].
Statistical Analysis
Statistical analysis was conducted by SPSS 11.0 software (SPSS, Chicago, IL) for Windows (Microsoft, Redmond, WA). Randomization was performed by a computer-generated algorithm. Data are presented as mean ± SD. Raw data were analyzed for normality of distribution. If not normally distributed, data were subjected to log transformation before analysis. Friedman analysis of variance (ANOVA) for repeated measures, followed by pair-wise comparisons (Wilcoxon signed ranks test with Bonferroni adjustment for the various comparisons of cytokines levels at each time point) was applied to detect changes in cytokine levels over time within the same group (placebo, statin, or off-pump). The nonparametric Kruskal-Wallis test was used to compare continuous data among the three arms. Proportions were compared by Fisher exact test when the expected frequency was less than five, otherwise the 
2 test (Yates’ corrected) was applied. A probability value of p < 0.05 was considered significant.
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Results |
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, IL-6, and IL-8 were significant over time within groups compared with the baseline values. Compared with the initial values, IL-8 levels peaked at 4 hours postoperatively, showing a continuous decrease during the first 72 hours postoperatively (Fig 1A). A significant difference between groups in the levels of IL-8 was found with repeated-measures ANOVA, and the IL-8 level was significantly higher only at 4 hours postoperatively in the placebo group compared with the statin and the OP groups (p < 0.01).
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levels increased progressively in all groups to reach a peak at 4 hours, with a slight return toward baseline values at 72 hours (Fig 1C). When analyzed by repeated-measures ANOVA, however, the only significant difference was found between the placebo and OP groups at 4 hours postoperatively.
Apoptosis
The mean percentage of apoptotic neutrophils was significantly higher in the simvastatin group compared with the placebo group at 12 hours (37.4% ± 12.2% versus 23.1 ± 8.1%; p < 0.05) and 24 hours (50.1% ± 14.3% versus 33.2% ± 9.8%, p < 0.01) of culturing (Fig 2A). We observed, however, that the prevalence of apoptosis was significantly higher among cells obtained from the OP patients compared with CPB patients independently of treatment (24 hours, p < 0.001 versus placebo and p < 0.05 versus statin), which is consistent with our previous findings [10].
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Fas and FasLigand Expression
Expression of Fas and Fas-L on neutrophil membrane was evaluated in a subset of patients from the CPB (n = 10 in each treatment group) and OP (n = 10) groups. Western blot analysis showed no significant difference in the expression of these proteins compared with controls.
ICE and CPP32 Expression
Expression of the proapoptotic caspases ICE and CPP32 was evaluated in a subset of patients from the CPB (n = 10 in each treatment group) and OP (n = 10) groups. Western blot analysis showed no significant difference in the expression of these proteins compared with controls (n = 10).
Neutrophil Activation
The values of NBT scores are shown in Fig 3A. Percentages of NBT-positive cells were high in all groups after 8 hours of culturing, when they reached the peak values, and then the values decreased in a linear fashion in all groups until 24 hours of culturing. Of interest was that during the entire examination period, the values of the NBT scores were significantly higher in the placebo group compared with the OP group (p < 0.01), whereas no significant differences were found between the OP and statin groups. At both 8 and 12 hours of culturing, the NBT score was significantly higher in the placebo group compared with the statin group (p < 0.05).
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Comment |
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Several past studies have demonstrated that the delay in neutrophil apoptosis is strictly linked to the increased plasma concentrations of IL-6 and IL-8 [16, 17]. In a previous study [13], we reported that deprivation of IL-6 and IL-8 from serum obtained from patients undergoing CPB completely abolished its antiapoptotic properties on cultured neutrophils. In the present report, we observed a significant increase of plasma cytokine levels in both OP and CPB groups over the entire time course of the study, with peak values in the postoperative period significantly higher in CPB patients. It is of interest that in these latter patients, peak levels of both IL6 and IL8 were significantly lower in the statin-treated group compared with the placebo group. These results are in keeping with those of two previous studies from our group showing that pretreatment with atorvastatin significantly reduced cytokine release and neutrophil adhesion to the venous endothelium after CABG with CPB [18, 19].
Activated neutrophils are the main source of proinflammatory cytokines in the systemic inflammatory syndrome; IL-6 and IL-8 facilitate survival of neutrophils by inhibiting apoptosis [20]. Hence, the system tends to enter a positive-feedback loop between prolonged survival and release of proinflammatory cytokines, with ongoing tissue and organ damage. It is therefore possible to speculate that the increase in the apoptotic rate of neutrophils as observed in the simvastatin group can be attributed to two processes: (1) significant reduction in availability of proinflammatory mediators, and (2) functional depression of neutrophils themselves. These processes seem to be independent and are likely to contribute separately to a more effective control of systemic inflammatory reaction after surgery with CPB.
Statins are among the most used and versatile cardiovascular drugs. Clinical trials have shown that they notably reduce cardiovascular morbidity and mortality in subjects with and without established coronary artery disease and improve cardiovascular outcome after CABG, irrespective of their efficacy at lowering cholesterol levels [14, 21, 22].
The use of simvastatin for cardioprotection in myocardial ischemia–reperfusion injury has been already proposed by Zheng and Hu [23]. They observed that in the isolated rat heart subjected to experimental ischemia–reperfusion, the acute infusion of simvastatin at a dose 100 µmol/L or less significantly ameliorated cardiac functional parameters, without alterations in heart rate and coronary blood flow. This occurred even in absence of neutrophils in the myocardial perfusate, and thus confirming a direct protective effect exerted by simvastatin.
Kaneider and colleagues [12] reported that cerivastatin reduced transendothelial migration and favored apoptosis in leukocytes at the site of atherosclerotic plaques. Because atherosclerosis is considered an inflammatory disease, these authors speculate that such effects of cerivastatin might be appreciable both at the myocardial and systemic level even in larger inflammatory reactions as it occurs after ischemia–reperfusion.
Finally, in a past study with an experimental model of hypoxia-reoxygenation [18], we demonstrated that pretreatment with simvastatin resulted in a significant downregulation both of P-selectin expression on endothelial cells and CD18 on stimulated neutrophils by an nitric oxide–mediated mechanism.
The autocrine interactions between Fas and Fas-L on the neutrophil membrane have been demonstrated to play an important role in the induction of apoptosis in human PMN cells, which constitutively express both Fas and Fas-L [5, 24]. In this study, we evaluated the expression of PMN Fas and Fas-L, but we could not find any significant difference in the expression of these receptors on PMN cells from the statin or placebo groups compared with PMN cells in the control group. In this context, our findings are in keeping with the report by Jimenez and colleagues [16] that showed no changes in the Fas expression of PMN cells in systemic inflammatory syndrome stress and with the observation by Liles and Klebanoff [5] showing that the incubation of PMN cells with IL-8, IL-6, and TNF- in vitro had no influence on the Fas expression. It is therefore possible to speculate that the Fas/Fas-L pathway is not the molecular mechanism by which simvastatin exerts its proapoptotic activity in neutrophils.
Fas and tumor necrosis factor receptor 1 signal for apoptotic cell death by downstream activation of proteins of the ICE family of proteases, including ICE and CPP32 [4, 25]. Caspases are the killer proteases that are activated during apoptosis. They are synthesized as inactive proforms and are activated in constitutive and Fas/apolipoprotein 1 (APO-1)–mediated apoptosis. In a separate set of experiments, we examined whether reduced PMN cell apoptosis induced by coronary operation was the result of decreased caspase activity. We found a considerable less caspase activity, as determined by DEVD-AMC cleavage, in freshly isolated neutrophils from the placebo group compared with the control patients. A decreased caspase activity was also found in neutrophils from the statin group compared with controls, but the difference failed to reach statistical significance.
Considering these results, we can hypothesize that (1) the increase of caspase 3 activity after simvastatin treatment is simply the consequence of the reduced plasma concentration of both IL-8 and IL-6, or (2) there is a direct activation of caspases by simvastatin. The reports of Blanco-Colio and colleagues [11] are in keeping with this latter hypothesis. These authors exposed exponentially growing venous smooth muscle cells to statins (100 µmol/L) and observed that caspase 9, the first caspase of the mitochondrial pathway, was cleaved during apoptosis induced by simvastatin in a time-dependent manner.
Neutrophils obtained from patients operated on off-pump displayed significantly higher apoptotic rates and lower indices of activation compared with the simvastatin group, which confirms the superior biocompatibility of off-pump surgery. Because the control of survival of neutrophils by simvastatin could favorably affect the acute inflammatory response after CPB, simvastatin might be proposed for routine pretreatment of patients scheduled for elective cardiac surgery, even for those free from traditional indications to statins. Future studies will clarify the clinical advantages of this approach and its cost-efficacy profile.
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