Ann Thorac Surg 2007;84:2000-2003. doi:10.1016/j.athoracsur.2007.07.034
© 2007 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Extracorporeal Circulation Activates Endothelial Nitric Oxide Synthase in Erythrocytes
Uwe M. Fischer, MDa,b,c,
Rüdiger Schindler, MSb,
Klara Brixius, PhDb,
Uwe Mehlhorn, MDc,d,
Wilhelm Bloch, MDb,*
a Michael E. DeBakey Institute, Texas A&M University, College Station, Texas
b Institute for Molecular Circulatory Physiology, German Sport University, Cologne, Germany
c University of Cologne, Cologne, Germany
d Department of Thoracic and Cardiovascular Surgery Johannes Gutenberg University, Mainz, Germany
Accepted for publication July 9, 2007.
* Address correspondence to Dr Bloch, Institute of Cardiovascular Research, Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Carl-Diem-Weg 6, Cologne, D-50933, Germany (Email: w.bloch{at}dshs-koeln.de).
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Abstract
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Background: Extracorporeal circulation used in cardiopulmonary bypass and hemodialysis is often associated with severe hypotension, which is an important predictor for mortality and morbidity. One pathophysiological hypothesis includes nitric oxide (NO) generation. Recently, a functional NO synthase (endothelial type NO synthase [eNOS]), was found to be expressed in human red blood cells. However, to date, activation of red blood cell eNOS has not been shown. We hypothesized that eNOS in circulating red blood cells might be activated during extracorporeal circulation and thus contribute to hypotension through vasodilation upon NO release.
Methods: We collected blood samples from 28 patients electively subjected to cardiac surgery during cardiopulmonary bypass (0, 20, 40, 60, and 80 minutes of cardiopulmonary bypass; taken for routine blood gas analyses). Red blood cells were immunohistochemically stained against activated eNOS. The degree of activation was assessed by television densitometry (gray units).
Results: We found significant red blood cell eNOS activation during cardiopulmonary bypass in a time dependent fashion. The eNOS activation occurred by dissociation of the enzyme from the cellular membrane into the cytosol (translocation). The correlation between activated eNOS and bypass duration was highly significant.
Conclusions: Red blood cells might contribute to hypotension through vasodilation upon NO release during extracorporeal circulation and could serve as new therapeutic targets in clinical practice.
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Introduction
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Extracorporeal circulation used in cardiopulmonary bypass and hemodialysis is often associated with severe hypotension [1, 2] as an important predictor for mortality and morbidity [3, 4]. Despite intensive research into this phenomenon the underlying pathophysiology is still poorly understood.
One proposed mechanism involves nitric oxide (NO), which plays an important role in regulation of blood pressure and blood flow. Nitric oxide is produced by three known synthases: the endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) NO synthase. In vascular wall, however, most of the bioavailable NO is believed to derive from eNOS. The intracellular signal mediation and mechanisms of eNOS activation in endothelial cells in the vascular system have been well described. Thus, eNOS activation can be accomplished by (1) dissociation of the enzyme from cellular membrane into the cytosol (translocation) and (2) an Akt-kinase–dependent serine eNOS phosphorylation in position 1177 (eNOSSer1177).
During extracorporeal circulation, shear-stress mediated platelet activation was shown to trigger serotonin-mediated nitric oxide release resulting in vasodilation [1, 5]. In addition, red blood cells (RBC) have been identified to reversibly bind, transport, and release NO within the cardiovascular system. In addition to early reports on NOS resident in RBC [6, 7], Kleinbongard and colleagues [8] could recently demonstrate that RBC from humans express an active and functional endothelial type NOS (eNOS), which is localized in the RBC plasma membrane and cytoplasm.
Hence, we hypothesized that eNOS in circulating RBC might be activated during extracorporeal circulation and thus contribute to hypotension through vasodilation upon NO release.
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Patients and Methods
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After approval from the local Ethics Committee and obtained written consent 28 patients electively undergoing cardiac surgery were included in the study (intraoperative variables are shown in Table 1). In all patients, a roller pump heart-lung machine with continuous perfusion (CAPS; Stoeckert/Sorin Group Germany, Munich, Germany) was used. The oxygenators and the bypass sets (Jostra Quadrox, Maquet Cardiopulmonary AG, Hirrlingen, Germany; Cobe Duo, Cobe Cardiovascular Inc, Arvata, CO; EOS 905, Dideco, Sorin Group, Arvada, CO) used were noncoated, and the bypass priming consisted of Ringer’s solution (1 L), hetastarch (1 L), bicarbonate (50 mL), and heparin (2000 IE). As cardioplegic solutions, we used Bretschneider’s solution (HTK, Custodiol; Köhler Chemie, Alsbach-Hähnlein, Germany) in heart valve surgery and warm intermittent blood cardioplegia according to Calafiore in coronary artery bypass graft surgery patients. Blood suctioned from the pericardium was scavenged and returned to the bypass reservoir. In all patients, anticoagulants such as aspirin were paused 8 days before surgery. All coronary artery bypass graft surgery patients received beta-blockers as wells as nitrates before surgery. We collected blood samples at different time points during cardiopulmonary bypass (0, 20, 40, 60, and 80 minutes of cardiopulmonary bypass; taken for routine blood gas analyses). Blood samples were centrifuged, and the plasma was removed.
For immunohistochemistry, erythrocytes were fixed with 4% paraformaldehyde in 0.1M phosphate-buffered saline (PBS) for 30 minutes. After several washing steps in 0.1M PBS, erythrocytes were stroked on glass and heat fixed. Thereafter, they were washed in 0.1M tris-buffered saline, permeabilized for 30 minutes with 0.1% trypsin, placed in a solution of 2% hydrogen peroxide and 80% methanol PBS for 20 minutes, and treated with 3% milk powder in 0.1M TBS for 30 minutes at room temperature. Incubation with the primary antibody was performed for 40 minutes in a TBS solution containing 0.3% milk powder, 0.03% Tween 20, and the primary polyclonal rabbit antibody against eNOS (1:1000; Biomol, Hamburg, Germany), which has been shown to be specific for the translocated active form of eNOS by Bloch and colleagues [9].
After rinsing with TBS, the sections were incubated with the secondary goat–anti-rabbit antibody (Dako, Glostrup, Denmark) at a dilution of 1:400 for 30 minutes. For negative controls, erythrocytes without primary antibody incubation were used.
A streptavidin-horseradish-peroxidase conjugate (Amersham, Buckinghamshire, England) was applied as a detection system (1:150 dilution) for 30 minutes. The staining was developed with 3,3’-diaminobenzidine-tetrahydrochloride solution in 0.1M PBS. For analysis only, erythrocytes incubated in the same immunostaining under identical conditions were compared.
For intensity analysis of immunostaining in erythrocytes, we measured the gray values (television densitometry) of 30 RBC from three randomly selected areas of each slice. The intensity of immunostaining was reported as the mean of measured cardiomyocyte gray value minus background gray value. The background gray value was measured at a cell-free area of the slice. For staining intensity detection, a Leica microscope coupled to a CCD-camera (DXC-1850P; Sony, Cologne, Germany) was used, and the analysis was performed using the software Image Version 1.33 (National Institutes of Health, Bethesda, Maryland).
All data presented are mean ± SEM. Data were analyzed for statistical significance on a level of alpha = 5% using analysis of variance with Tukey’s test for post-hoc comparison as implemented in the software package SPSS for Windows, version 10.0 (SPSS, Chicago, Illinois).
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Results
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Figure 1A depicts activated eNOS as measured by an antibody against translocated and activated eNOS [9] assessed by TV densitometry (gray units). The graph shows a time-dependent increase in eNOS activation in a linear fashion. The correlation between activated eNOS and cardiopulmonary bypass duration shown in Figure 1B is highly significant (r = 0.98; p = 0.0008). Representative immunohistochemical stainings for each time point are shown in Figure 2.
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Fig 1. (A) Analysis of endothelial type nitric oxide synthase (eNOS) activation by means of television densitometry (gray units) reveals a linear increase in eNOS activation as compared with baseline levels (0 minutes). Analyses were performed at 0, 20, 40, 60, and 80 minutes of cardiopulmonary bypass (CPB) perfusion. Values are expressed as mean ± SEM. *p < 0.05. (B) The correlation between eNOS activity and cardiopulmonary bypass duration is highly significant (r = 0.98; p = 0.0008).
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Fig 2. Immunohistochemical stainings of erythrocyte activated endothelial type nitric oxide synthase (eNOS). Activation of eNOS in red blood cells is shown at different time points during cardipulmonary bypass. Staining intensity increased in a time-dependent fashion. (A) Immunhistochemistry control (without primary antibody) is shown. (B–F) Red blood cell staining is shown at 0, 20, 40, 60, and 80 minutes of cardiopulmonary bypass, respectively. Bar = 10 µm. Magnification for all images 400x.
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Comment
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Our data show significant eNOS activation in circulating RBC during cardiopulmonary bypass in a time-dependent fashion. Activated eNOS was highly correlated with cardiopulmonary bypass duration.
As one cause of severe hypotension associated with the use of extracorporeal systems both in cardiac surgery and hemodialysis, platelet aggregation with subsequent release of serotonin and adenosine diphosphate has been identified. Serotonin in turn not only has vasodilating properties, it also stimulates endothelium-dependent release of NO. Elevated levels of NO during extracorporeal circulation have been reported by several authors [10, 11]. Thus, increased NO release seems to play a major role in the etiology of extracorporeal circulation–associated hypotension.
Circulating red blood cells might also serve as addditional source of NO release, as only recently demonstrated by Kleinbongard and colleagues [8] in addition to previous studies [6, 7] have reported functional eNOS expression in RBC. We found increased RBC eNOS activity during cardiopulmonary bypass assessed by eNOS translocation, which has been identified as one intracellular mechanism for eNOS activation [9, 12]. The RBC eNOS activation during extracorporeal circulation appears as plausible mechanism, as extracorporeal circulation not only activates platelets but also results in shear-stress–mediated eNOS activation in vascular endothelium [13]. Furthermore, Cheng and associates [14] have recently shown the effect of shear stress on eNOS distribution in endothelial cells, which might also apply to circulating RBC. In addition, Pott and coworkers [15] have demonstrated eNOS translocation rather than phosphorylation to be the dominant mechanism for NO generation in atrial myocardium.
Considering these findings in combination with our results, we conclude that extracorporeal circulation activates RBC eNOS through translocation, and thus might contribute to extracorporeal circulation–associated hypotension through subsequent NO release. As we only found a time-dependent activity increase independent of the operative procedure and associated variables such as different cardioplegic strategies, we attribute RBC eNOS activation mainly to extracorporeal circulation–associated mechanical stress.
Our data do not allow us to speculate to what extent RBC eNOS contributes to NO generation during extracorporeal circulation. However, the results of our study build the rationale for future studies on the activation of RBC eNOS, the associated NO generation, and the potential role in vascular tone regulation, not only during extracorporeal circulation.
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References
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Abstract
Introduction
