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CARDIOVASCULAR ANESTHESIA

Endothelial Ca²⁺ Signal Transduction Is Altered by Postoperative Serum from Patients Undergoing Coronary Surgery with Cardiopulmonary Bypass

Department of Anesthesiology and Intensive Care Medicine, Charitè-Universitätsmedizin Berlin, Berlin, Germany

Address correspondence to Thomas Volk, MD, Klinik für Anästhesiologie und Operative Intensivmedizin, Charité Universitätsmedizin Berlin, Campus Mitte. Schumannstr. 20/21, 10117 Berlin, Germany. Address e-mail to thomas.volk{at}charite.de.

	Abstract

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Endothelial dysfunction after surgery may be caused by alterations in the intracellular signaling properties of endothelial cells. Functional alterations are believed to be systemic and dependent on the amount of invasiveness. This led us to assume that there would be a mediator in the blood. Therefore, we investigated the influence of perioperative serum obtained from patients undergoing highly invasive surgical interventions (cardiac surgery [CS] with cardiopulmonary bypass) and less invasiveness (total joint arthroplasty [TJA]) on endothelial single cell Ca²⁺ responses. Aortic endothelial cells were incubated with preoperative and postoperative serum samples from 26 patients undergoing CS and from 15 patients undergoing TJA. Adenosine triphosphate (100 µM)-induced alterations in FURA-2 fluorescence was used to measure intracellular Ca²⁺ in single cells. In CS samples the induced [Ca²⁺]_i signals were enhanced by postoperative serum (peak levels: 96 ± 41 FU versus 116 ± 45 FU; P < 0.05). These postoperative enhancements were absent in TJA patients serum. Preincubation of CS samples with nifedipine to block voltage gated Ca²⁺ channels did not alter this effect, but the absence of extracellular Ca²⁺ abolished the increased response from postoperative CS serum exposure. Ca²⁺ entry probed with Mn²⁺ quenching was increased in endothelial cells exposed with postoperative CS serum and Ca²⁺ entry correlated with postoperative circulating interleukin-6 levels (P < 0.007). Endothelial functional alterations after CS with cardiopulmonary bypass are attributable, in part, to systemic factors present in serum that lead to specific endothelial enhanced Ca²⁺-signaling. This enhancement can be separated in vitro as an increased Ca²⁺ entry not present in serum from patients recovering from TJA.

	Introduction

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Endothelial cells are the first layer of the border between blood circulation and tissue. Their functional importance in many biological aspects of homeostasis is remarkable. Pathogenetically this cell type has been implicated to be involved in postoperative inflammatory reactions. Cardiac surgery (CS) involving cardiopulmonary bypass (CPB) is associated with a postoperative systemic inflammatory response (1–3), which may cause organ dysfunction and may lead to multiple organ failure. Even though their regulatory impact on permeability, leukocyte traffic, and blood supply to organs is significant, the functional state of endothelial cells is difficult to monitor clinically.

Alterations of endothelial functions can be investigated by measuring circulating markers of activation, such as PECAM-1, P-selectin, ICAM-1 or E-selectin (4,5), the quantification of endothelial-dependent vasodilation or in animal models (6). A direct approach to define endothelial dysfunction is possible by investigating endothelial cells in vitro. Blood components from patients undergoing CS activate endothelial cells (4,7). Whether altered signaling pathways in endothelial cells are involved in the development of a systemic inflammatory response syndrome (SIRS) has been only partially investigated (8).

Because cytosolic-free calcium concentration is crucial for the control and regulation of many endothelial cellular functions, we decided to focus our investigation on this messenger and hypothesized that soluble factors present in the serum of patients are mediating alterations in intracellular signaling pathways.

We also assumed that any postoperative alteration that we may find might be related to the amount of invasiveness or the amount of physiological derangement. CS involving CPB causes major alterations(cytokine secretion, both proinflammatory and antiinflammatory responses) in the physiological balance of many mediators. To compare results with less invasive surgery, in which SIRS would be less likely but which can still be regarded as major surgery (in contrast to minor surgery such as outpatient hernia repair), we chose patients undergoing total joint arthroplasty (TJA).

	Methods

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After approval of the local ethics committee, written informed consent was obtained from patients undergoing orthopedic surgery (Ref. No. 1454/2000) and CS (Ref. No. 1123/99), respectively. Inclusion criteria were age >18 yr and planned coronary or endoprosthetic surgery. Exclusion criteria were severe liver disease (Child B), pregnancy, insulin-dependent diabetes mellitus, or the presence of a systemic and chronic inflammatory disease. Patients undergoing CPB were anticoagulated with 350 U/kg heparin (Liquemin®; Hoffmann-La-Roche, Grenzach-Wyhlen, Switzerland) and additional boluses of 50 U/kg were given if necessary to maintain an activated clotting time of at least 480 s. All patients on CPB received aprotinin (Trasylol® 50,000 IU/kg; Bayer, Leverkusen, Germany). Routine CPB priming included hydroxyethyl starch 10% (500 mL, Fresenius Kabi, substitution grade 0.4–0.55, mean molecular weight 200,000), electrolyte solution (Thomaejonin® 500 mL; Delta Pharma GmbH, Pfullingen, Germany) and heparin (8000 U). CPB was performed under slightly hypothermic conditions (34.5°C–35.5°C) using a membrane oxygenator (Quadrox®; Jostra, Hirrlingen, Germany) and centrifugal pump flows (Bio-Medicus®, Medtronic, Indianapolis, IN) adjusted to 120% of the calculated cardiac index of 2 L/min/m²).

According to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) classification (9), SIRS was diagnosed if 2 or more of the following signs were found: body temperature abnormalities (>38°C or < 36°C), tachycardia (heart rate >90 bpm), tachypnea or hyperventilation (respiratory rate >20/min or Paco₂ < 32 mm Hg), or leukocytosis or leukopenia (white blood cell count >12 Gpt/L or <4 Gpt/L). SIRS classification was performed in all patients on the first postoperative day at 8.00 am considering the whole postoperative course.

Preoperative blood samples (T1) were withdrawn from venous blood immediately before skin incision and postoperative blood samples (T2) were collected 4 h after the completion of surgery. Serum was immediately stored in liquid nitrogen.

Human aortic endothelial cells (HAEC; Clonetics®, Cambrex, Nottingham, UK) were expanded and subcultured according to the manufacturer's instruction. For the experiments, cells were grown on glass slides to subconfluence. The incubations with perioperative serum samples were performed for 2 h with 20% of the indicated sample and 80% EGM-2 (a serum free endothelial growth medium; Clonetics) at 37°C.

Measurement of intracellular calcium concentration ((Ca²⁺)_i) was performed as previously described (8). Briefly, cells were loaded for 40–50 minutes at room temperature with the calcium sensitive probe FURA-2/am. (1 µM; Calbiochem, Darmstadt, Germany) dissolved in dimethylformamide (1 µM). [Ca²⁺]_i measurements were performed in a buffer (127 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 0.5 mM NaH₂PO₄, 10 mM D-glucose, pH 7.3, plus 1.8 mM CaCl₂ or 1.8 mM EGTA, respectively). Mn²⁺ quenching experiments were performed in a buffer consisting of 127 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM D-Glucose, 1 mM MnCl₂ and 5 mM HEPES at pH 7.4.

The Ca²⁺-concentration in the intracytoplasmic space is very small (50-100 nM) compared with the extracellular space or compared with Ca²⁺-stores within a cell (1-2 mM). FURA-2 can be trapped within the cytoplasm when the cell-permeable acetoxymethylester of FURA-2 (FURA-2/am) is cleaved by unspecific cytoplasmic esterases. The fluorescence of intracellular FURA then is related to changes in [Ca²⁺]_i. The addition of adenosine triphosphate (ATP) in the absence of extracellular Ca²⁺ activates the release of intracellular stores, and this release activates the store-operated calcium entry path. To mimic Ca²⁺ entering the cell Mn²⁺ can be added to the extracellular space. Mn²⁺ enters the cell through these Ca²⁺-channels when they are open. Mn²⁺ then competes with the Ca²⁺-binding sites of FURA and causes a decline in fluorescence. Thus the Mn²⁺ quenching of FURA fluorescence (the decline in fluorescence) was used to quantify Ca²⁺ entry.

For cell imaging, a digital analysis system (T.I.L.L.; Photonics, Planegg, Germany) connected to an inverted microscope (DMIRB; Leica, Germany) equipped with a Zeiss Fluor 1,3/40x oil objective was used. Excitation wavelengths (340 nm, 360 nm, 380 nm) were produced by a scanning monochromator and images of fluorescence passing a 395 nm dichroic mirror and a 520 nm band pass filter were recorded by a cooled slow scan CCD camera at indicated intervals. Intracellular (Ca²⁺) data are given as fluorescence units (FU) of the measured light intensity ratio of the 380 nm excitation divided by the 340 nm excitation after background subtraction. One glass slide was measured only once with at least 5 cells analyzable after addition of the stimulus. At least 10 glass slides (10 stimulations) were used for incubations of each time point for each individual patient.

ATP was purchased from Boehringer (Ingelheim, Germany). Cyclopiazonic acid (CPA) and FURA-2/am were obtained from Calbiochem (Darmstadt, Germany). MnCl₂, nifedipine and EGTA was purchased from Sigma (St. Louis, MO). CaCl₂, NaCl, NaHPO₄, glucose, MgCl₂, KCl, NaHCO₃ and DMSO was purchased from Merck (Darmstadt, Germany). All substances were of the highest available purity.

Our investigation was planned as a pilot study. From previous investigations we speculated that at least 70% of our patients would develop a SIRS and we also would expect that the majority would have increased levels of interleukin (IL)-6 (3). A relevant decrease in Ca²⁺-signal characteristics is in the range of 15%–20%. In the absence of previous data concerning intracellular Ca²⁺-alterations from ex vivo experiments in the perioperative setting we chose an arbitrary, but reasonable, number of 30 patients. Of these, 4 had to be excluded because the necessary amount of serum was not obtained or was lost during the process of preparation. None of the patients undergoing joint replacement surgery was expected to develop SIRS, and we thus chose to include only half as many patients in this group.

From each single cell, the peak (Ca²⁺)_i-response was calculated after addition of the respective stimulus. The area under the curve (AUC) was calculated by adding all measured FU values during a fixed time period of 50 s. Comparisons between preoperative and postoperative mean values (peak, AUC, absolute increases from individual baseline value to peak value) were done by using Student's t-test after testing for normality with the Kolmogorov-Smirnov test. Significant difference between the respective values was assumed at P < 0.05. If not otherwise stated, mean ± sd is given.

	Results

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Of 30 patients undergoing CS 4 had to be excluded because the necessary amount of serum was not obtained (n = 2) or was lost during the process of preparation (n = 2). The remaining 26 patients undergoing coronary artery bypass graft (CABG) surgery with CPB were similar in age and gender distribution compared to patients undergoing TJA (hip replacement, 12; knee replacement, 3). Patients undergoing CS had longer operation times and the incidence of a postoperative SIRS as defined by the ACCP/SCCM was more frequent in this population (Table 1).

Single cell [Ca²⁺]_i increased by 58 ± 32 FU after addition of ATP (100 µM) in endothelial cells exposed to preoperative serum from patients undergoing CS after 10 s and then declined briefly to remain at a constant level for the rest of the observation period (AUC: 4308 ± 1780; peak: 96 ± 41 FU). Endothelial cells incubated with serum from CS patients obtained postoperatively showed an increase in peak [Ca²⁺]_i levels after stimulation with ATP by 72 ± 42 FU (AUC: 5162 ± 1844 FU; peak: 116 ± 45 FU). Mean increases (P < 0.00001), peak levels (P < 0.0001), and AUC (P < 0.0001) were significantly higher in cells stimulated after postoperative CS serum exposure compared with preoperative CS serum exposure (Fig. 1A). ATP-induced [Ca²⁺]_i increase in endothelial cells incubated with preoperative serum from TJA patients was similar to preoperative CS patients ( 51 ± 29 FU; AUC: 4564 ± 1740; peak: 99 ± 44 FU). ATP stimulation of cells exposed to postoperative TJA serum caused increases of [Ca²⁺]_i by 54 ± 36 FU (AUC: 4635 ± 1860; peak: 100 ± 42 FU). Mean increases, peak levels, and AUC were not increased in cells stimulated after postoperative TJA serum exposure compared to preoperative TJA serum exposure (Fig. 1B).

View larger version (33K): [in this window] [in a new window]   Figure 1. [Ca2+]i changes in single endothelial cells stimulated with adenosine triphosphate (100 µM; arrow indicates the addition) after 2 h of preincubation with serum obtained preoperatively (black dots) and postoperatively (white dots). Data shown are mean ± sd from cardiac surgery patients (A; 412 cells were measured with preoperative serum and 427 cells were measured with postoperative serum) and orthopedic patients (B; 346 cells were measured with preoperative serum and 295 cells were measured with postoperative serum), respectively.

After preincubation of HAEC with nifedipine (1 µM) to block voltage-gated channels, ATP-induced [Ca²⁺]_i of cells incubated with preoperative CS serum increased by 45 ± 18 FU (AUC: 3600 ± 1265; peak: 84 ± 24). Nifedipine incubation to HAEC exposed to postoperative serum from CS patients increased [Ca²⁺]_i after ATP stimulation by 73 ± 12 FU. (AUC: 4605 ± 2012; peak: 108 ± 22 FU). In the presence of nifedipine mean increases (P < 0.001), peak levels (P < 0.01), and AUC (P < 0.01) were still significantly higher in cells stimulated after postoperative CS serum exposure compared with preoperative CS serum exposure. The postoperative serum effect of CS patients was still present after blockade of voltage-gated channels, leading us to suggest that voltage-gated channels are not involved. Nifedipine-treated HAEC exposed to preoperative TJA serum showed ATP-induced (Ca²⁺)_i-increases of 45 ± 14 FU (AUC: 3464 ± 1320; peak: 79 ± 21 FU), and incubation with postoperative serum from TJA patients showed ATP-induced increases of 51 ± 9 FU (AUC: 3757 ± 1614; peak: 86 ± 11 FU). No significant differences in increases, peak levels, or AUC compared to preoperative TJA incubations were found.

Removing Ca²⁺ from the buffer caused a lower signal in endothelial cells incubated with serum from all patients regardless of time and operation. ATP stimulation after serum incubation from preoperative CS led to increases by 45 ± 14 FU (AUC: 3258 ± 1522; peak: 78 ± 12 FU), and ATP stimulation after serum incubation from postoperative CS led to increases by 45 ± 13 FU (AUC: 3162 ± 1424; peak: 75 ± 15 FU). No significant differences in mean increase, mean peak, or mean AUC levels were found. Stimulation after preoperative TJA serum incubation caused increases by 50 ± 10 FU (AUC: 3411 ± 1534; peak: 81 ± 19 FU) and stimulation after postoperative TJA serum incubation led to increases by 50 ± 12 FU (AUC: 3333 ± 1211; peak: 79 ± 14 FU). No significant difference in mean increase, mean peak, or mean AUC levels were found. Under these conditions, an increased Ca²⁺ signal in endothelial cells exposed to serum from patients after CS was no longer present, indicating that serum from postoperative CS patients mediates an increased Ca²⁺ influx from the extracellular space. (Fig. 2).

View larger version (27K): [in this window] [in a new window]   Figure 2. [Ca2+]i changes in single endothelial cells in the absence of extracellular Ca2+ after 2 h of preincubation with serum obtained preoperatively (black dots) and postoperatively (white dots). Data shown are mean ± sd from cardiac surgery patients (A; 56 cells were measured after preoperative serum exposure and 61 cells were measured after postoperative serum exposure) and orthopedic patients (B; 55 cells were measured after preoperative serum exposure and 66 cells were measured after postoperative serum exposure), respectively. Arrow indicates the addition of adenosine triphosphate (100 µM).

There is a steep Ca²⁺ concentration gradient between the endoplasmic reticulum and the cytoplasm. Therefore, Ca²⁺ has to be actively and continuously pumped out of the cytoplasm to compensate for a continuous leak. CPA (10 µM) blocks the reuptake of Ca²⁺ into the endoplasmic reticulum, leading to an emptying of the endoplasmic reticulum. A difference in the releasing capacity induced by CPA may show a difference in the loading conditions of the endoplasmic reticulum. The increase in fluorescence on application of CPA in the absence of extracellular Ca²⁺ should therefore correlate with the amount of Ca²⁺ released from intracellular stores. Endothelial cells incubated with preoperative CS serum under these conditions showed a CPA-induced mean [Ca²⁺]_i increase of 12 ± 3 FU (peak: 39 ± 3 FU; AUC: 1745 ± 952). Incubation with postoperative CS serum showed a CPA-induced mean [Ca²⁺]_i increase of 16 ± 4 FU (peak: 49 ± 6 FU; AUC: 2116 ± 1030). In the presence of CPA without extracellular Ca²⁺ mean increases (P < 0.001), peak levels (P < 0.001), and AUC (P < 0.001) still were significantly higher in cells stimulated after postoperative CS serum exposure compared with preoperative CS serum exposure. Serum obtained from TJA patients showed no significant difference in CPA-induced Ca²⁺ release characteristics (increase, peak, and AUC) between preoperative and postoperative samples (preoperative TJA increase: 14 ± 3 FU; peak: 42 ± 5 FU; AUC: 1862 ± 1254; postoperative TJA increase: 11 ± 4 FU; peak: 39 ± 4 FU; AUC: 1809 ± 1310). This experiment may indicate a slightly increased emptying of the endoplasmic reticulum in the early signaling phase when postoperative CS serum is used. Endoplasmic release of Ca²⁺ may activate the store-operated Ca²⁺-channel within the cell membrane. The activation of these channels are most important for non-excitable cells and usually lead to larger and prolonged cytoplasmic Ca²⁺ increases.

Mn²⁺-quenching of FURA-2 fluorescence was used to quantify the rate of Ca²⁺-entry. The decline of FURA-2 fluorescence in endothelial cells incubated with serum from postoperative CS patients was significantly steeper (slope of linear regression fit: –0.36 ± 0.15) than the decline in endothelial cells incubated with serum from preoperative CS patients (slope of linear regression fit: –0.16 ± 0.10; P < 0.01; Fig. 3A). Again there was no difference in steepness of Mn²⁺-quenching in HAEC incubated with serum from TJA patients when preoperative (slope of linear regression fit: –0.20 ± 0.14) and postoperative (slope of linear regression fit: –0.23 ± 0.09) serum incubations were compared (Fig. 3B).

View larger version (25K): [in this window] [in a new window]   Figure 3. Decreases in FURA fluorescence signal after addition of Mn2+ in adenosine triphosphate (100 µM)-stimulated endothelial cells. Single endothelial cells in the absence of extracellular Ca2+ after 2 h of preincubation with serum obtained preoperatively (black dots) and postoperatively (white dots) are shown. Data shown are mean ± sd from cardiac surgery patients (A; 62 cells measured after preoperative serum incubation and 53 cells were measured after postoperative serum incubation) and orthopedic patients (B; 52 cells were measured after preoperative serum incubation and 58 cells were measured after postoperative serum incubation), respectively. The arrow indicates the addition of Mn2+ (100 µM).

IL-6 has been widely used as a marker of the intensity of an inflammatory response. IL-6 levels in both surgical groups significantly increased in the postoperative period compared with preoperative values (P < 0.001 each; Table 1). Postoperative IL-6 levels in patients undergoing CS also were significantly higher than in patients undergoing TJA (P < 0.001; Table 1). Using linear regression analysis, a significant correlation was found between postoperative IL-6 levels and the stimulated mean Ca²⁺ response in endothelial cells (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Figure 4. Correlation between postoperative serum concentration of interleukin-6 and adenosine triphosphate-induced [Ca2+]i changes in single endothelial cells. Cells were preincubated for 2 h with serum obtained postoperatively. Data shown are from cardiac surgery patients (black dots) and orthopedic patients (white dots), respectively.

	Discussion

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Many authors have suggested that endothelial dysfunction may contribute to postoperative complications of patients undergoing CABG with CPB. The present investigation shows that postoperative serum from these patients causes an enhanced stimulated Ca²⁺-signaling cascade in endothelial cells ex vivo compared to preoperative serum exposure. Less invasive surgery (joint arthroplasty) does not produce this systemically mediated ex vivo effect. By pharmacologically separating signaling pathways, we found that the store-operated Ca²⁺ entry contributes most likely to this enhanced Ca²⁺-signaling.

Agonist-stimulated increase of [Ca²⁺]_i in EC may occur by Ca²⁺ influx through plasmalemmal cation channels, by IP₃ sensitive release of Ca²⁺ from intracellular stores, and by capacitative calcium entry, which is a mechanism that involves Ca²⁺ influx from the extracellular space triggered by Ca²⁺ release from intracellular stores (10). ATP binds surface P₂ receptors coupled to G-protein activated IP₃ formation. ATP has been shown to be actively released and contributes to vascular tone regulation. Like other purine agonists, it can be used as a signaling probe (11), but certainly no agonist is universally representative for G-protein coupled receptor binding.

Enhanced stimulated [Ca²⁺]_i peak increase in HAEC incubated with serum obtained from patients after CS was mainly attributable to enhanced Ca²⁺ influx from the extracellular space because it was not influenced by the voltage-gated channel blocker nifedipine, abolished in the absence of extracellular Ca²⁺, slightly present when CPA was used, and clearly active when Mn²⁺-quenching of FURA fluorescence was used.

Cultured endothelial cell are usually devoid of voltage-gated channels, but HAEC may have nifedipine-responsive channels after platelet activating factor stimulation when freshly isolated first passage cells were used (12). As the increased response to ATP persisted in the presence of nifedipine, it may reflect the absence of L-type voltage gated channels in the HAECs used in the present investigation.

CPA acts as a reversible inhibitor of the Ca²⁺-ATPase that is responsible for the sequestration of Ca²⁺ from the cytoplasm into the endoplasmic reticulum, where most of the intracellular Ca²⁺ is stored. The increase in fluorescence on application of CPA should therefore correlate with the amount of Ca²⁺ released from intracellular compartments. An enhanced stimulated [Ca²⁺]_i for HAEC incubated with postoperative serum from CS patients may either reflect a larger amount of Ca²⁺ stored or an accelerated release. A slightly increased release was found, indicating that endoplasmic stores may contain more Ca²⁺ after incubation with postoperative CS serum.

The release of Ca²⁺ from the endoplasmic reticulum activates Ca²⁺ entry through the cell membrane, giving rise to the calcium release-activated calcium current. The Ca²⁺ concentration in the intracytoplasmic space is very small (50–100 nM) compared with the extracellular space or compared with Ca²⁺ stores within a cell (1–2 mM). The addition of ATP in the absence of extracellular Ca²⁺ activates the release of intracellular stores and this release activates the store-operated calcium entry path. To mimic Ca²⁺ entering the cell by these store-activated Ca²⁺-channels, Mn²⁺ is added to the extracellular space, which enters the cell through these Ca²⁺ channels when they are open. Mn²⁺ then reacts with FURA and causes a decline in fluorescence (quenching). The steeper the decline, the faster Mn²⁺ enters the cell (instead of Ca²⁺). Our experiments using Mn²⁺ quenching of FURA-2 fluorescence showed that postoperative serum from CS patients had an increased Mn²⁺ entry that is functionally equivalent to an activated Ca²⁺ entry from the extracellular space into the cytoplasm.

The functional significance of our findings lies in the many Ca²⁺-dependent pathways of endothelial cell activation assumed to be present postoperatively. This activated state has been deduced from studies showing increases in endothelial permeability or endothelial adhesion molecules released postoperatively into the circulation. An increased Ca²⁺ entry may explain reports of increased Ca²⁺-regulated endothelial surface expression of adhesion molecules by the exposure of postoperative serum from CS patients (4) or the redistribution of VEe-cadherin reported from coculture experiments (7). Moreover, only postoperative serum from patients undergoing CABG surgery with CPB exposed to endothelial cells ex vivo has been shown to induce apoptosis, a process that is closely linked to Ca²⁺- homeostasis (13).

Endothelial responses to stimuli have a large variability (14). Published data on significantly altered peak Ca²⁺ responses are in the range of 15%–20%. Translating these signals into an effect size for functional alterations may well be in this range, as has been shown for apoptosis (15) or adhesion molecule expression (16).

CS involving CPB techniques lead to greater postoperative inflammatory reactions compared with less invasive surgery such as hip or knee replacement. There are many differences between these procedures, of which the CPB may have a leading role. However, patients with off-pump surgery were not included in the present investigation to evaluate the specific influence of CPB. Postoperative inflammatory reactions can be quantified by circulating IL-6 levels. This cytokine has been shown to be the responsible myocardial depressant factor in sepsis patients (17). The clear correlation between IL-6 levels and the activation of Ca²⁺ entry highlights the coincidence for the present investigation but does not indicate causality. VEGF, an angiogenic growth factor, has been reported to increase after CS (18) and signals an increase in permeability via [Ca²⁺]_i in a subset of endothelial cells (19,20). Candidate mediators could be removed from the serum either chemically or by antibodies to define the nature of the presumed mediator. Patients in the CS group also had a higher degree of postoperative circulatory disturbance reflected by more vasoactive drug treatment. Whether altered endothelial Ca²⁺ signaling is responsible for this disturbance can only be speculated on. Intraoperatively, patients did not receive more antihypertensive therapy than vasopressors. We thus cannot speculate on a preferential drug effect that could be responsible for an altered endothelial signaling.

One limitation of the current approach is that ex vivo experiments may not accurately reflect continuing processes in vivo, as endothelial cells may have more time to react in an altered environment. Patients undergoing CS had more comorbidities and more chronic medication that potentially could have influenced the ex vivo results but had only minor influence on the preoperative inducible Ca²⁺ response. Whether statins or ß-blockers alter the postoperative inflammatory response and thereby influence endothelial Ca²⁺ signaling remains to be shown. The fact that we used serum instead of plasma may neglect that coagulation factors bind endothelial cells and also potentially alter their function. The observer was not blinded as to the type of serum and therefore potentially may have biased the selection of cells used for measurements.

Numerous investigations have shown that many inflammatory markers are transiently increased after CABG surgery with CPB, but the biological consequences are unclear. The significance of patients' postoperative vascular function (e.g., endothelial-dependent relaxation or permeability measurements) should be investigated. Endothelial dysfunction can be expected to depend on store-operated Ca²⁺ entry mechanisms (e.g., nitric oxide secretion, endothelial cytokine secretion, endothelial apoptosis) on the basis of present findings.

In summary, we conclude that postoperative circulating mediators present in serum alters endothelial cell signaling functions ex vivo. This alteration may be the result of the aggregate activity of all possible mediators (e.g., proinflammatory and antiinflammatory). The altered signaling cascade in endothelial cells by postoperative serum is characterized by an activated Ca²⁺ entry. Whether a pharmacological inhibition of this pathway improves the sequelae of postoperative inflammatory reactions remains to be shown.

	Footnotes

 
Accepted for publication January 31, 2006.

Reprints will not be available from the author.

Supported, in part, by the Forschungsförderung Charité, Humboldt University Berlin.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999