INTRODUCTION

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Cardiopulmonary bypass (CPB) in infants and children often results in pulmonary hypertension (1), a potential source of considerable morbidity and even mortality. It has been suggested that the mechanisms underlying this increase in pulmonary vascular resistance involve the widespread inflammatory reaction to CPB. Several studies have identified and measured high levels of various circulating inflammatory mediators (2- 6), and have also demonstrated leukocyte activation during CPB (4, 7). Recent work has focused on the role of polymorphonuclear neutrophils (PMNs) in endothelium injury following CPB (5, 8). The adherence of PMNs to the pulmonary endothelium is the crucial early step in both endothelial injury and subsequent transendothelial migration (5). Activated PMNs are stimulated both to adhere to the endothelium and to release reactive oxygen species (9) and cytotoxic enzymes (10). That pulmonary vascular endothelium dysfunction is involved in the pulmonary hypertensive response to CPB has been established in recent years (11, 12). The demonstration of capillary leakage and tissue injury has also provided indirect evidence of pulmonary endothelial injury. Earlier studies indicated that the inflammatory response to CPB was induced by the contact of blood with nonphysiologic surfaces. In addition, we recently demonstrated that pulmonary vascular endothelial cells undergo ischemia-reperfusion injury during total CPB, and suggested that this may further aggravate the inflammatory response and subsequent lung endothelium damage (13). Our aim was to separate out the effects of membrane oxygenator- induced activation of leukocytes from lung ischemia-reperfusion on the function and viability of the pulmonary and systemic endothelia in neonatal piglets. Interactions between leukocytes harvested after various times of CPB and cultivated endothelial cells were investigated and related to in vivo leukocyte sequestration in the pulmonary and systemic microvascular beds. In addition, viability of aortic and pulmonary artery endothelial cells and endothelial vascular reactivity in isolated femoral and pulmonary arterial rings were assessed before, during and after total CPB, and the results were correlated with in vivo systemic and pulmonary hemodynamics.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Neonatal piglets (n = 18; mean age, 7 ± 2 d; mean weight, 3.2 ± 0.3 kg) were purchased from a local farmer. All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. They were fasted for 12 h before the operation, sedated with intramuscular ketamine (250 mg), and anesthetized with pentobarbital sodium (15 mg/kg intravenously). The animals were then intubated, and general anesthesia was maintained using 60% N₂O₂/40% O₂ with intermittent positive pressure ventilation (MMS 107 ventilator; Radiometer, Paris, France) at 40 breaths/min with a tidal volume of 15 ml/kg. The animals were placed on their backs, and the femoral vessels on both sides were isolated. After right femoral artery cannulation, the ventilatory rate and tidal volume were adjusted to obtain normal pH and PCO₂ values, as determined by arterial blood gas measurement (ABL2-radiometer; Copenhagen, Denmark). After midline sternotomy and pericardiotomy, the ductus was ligated and the great vessels were controlled. A pulmonary artery catheter (Intracath, New York, NY) was introduced into the main pulmonary artery through the right ventricular infundibulum, and cardiac output was recorded using a Doppler probe placed around the pulmonary artery trunk (Model T106 M; Transonic Systems, Ithaca, NY). The central venous and left atrial pressures were monitored after placement of indwelling catheters in the right and left atria. Pressures were recorded using a P23 ID Statham pressure transducer (Statham, Ballainvilliers, France). After heparin administration (2.5 mg/kg), CPB was instituted between the right atrium and the ascending aorta. The bypass circuit consisted of a cardiotomy reservoir and a membrane oxygenator (Dideco, Gagny, France), a heat exchanger, and a roller pump. No arterial filter was used. The circuit was primed (400 ml) with fresh whole blood obtained from a donor pig on the day of surgery. Within the first 5 min of CPB, a vent was introduced through the left ventricular apex. Mean systemic arterial pressure was maintained at pre-CPB values with a mean flow of 500 ml/min. Once the steady state under CPB was achieved, ventilation was stopped and cooling to 28° C was started. The pulmonary artery trunk was cross-clamped to avoid anterograde flow to the lungs, and CPB was continued for 90 min without aortic cross-clamping. During CPB, collateral pulmonary venous blood flow was measured by collecting the blood draining from the left ventricle into an open reservoir, and expressed in milliliters per minute. After this period of lung ischemia, mechanical ventilation was restarted and the piglets were weaned from CPB. They were killed 1 h later. Throughout the experimentation, central, myocardial, and lung temperatures were monitored. Biopsy specimens for myeloperoxidase activity measurements in lungs and skeletal muscles were obtained before CPB; at 30, 60, and 90 min of CPB; and 30 and 60 min after CPB. Left femoral artery aorta and pulmonary arteries beyond the second-generation branch were dissected for evaluation of vascular reactivity or cultured endothelial cell study.

Hemodynamic and Arterial Blood Gas Measurements

Hemodynamic parameters were measured before CPB initiation and at 30-min intervals after CPB discontinuation. Cardiac output was determined as the pulmonary blood flow (pa in liters per minute). The cardiac index (CI), systemic vascular resistance index (SVRI), and pulmonary vascular resistance index (PVRI) were calculated as follows:

(1)

(2)

(3)

where MAP = mean systemic arterial pressure, RAP = mean right atrial pressure, PAP = mean pulmonary arterial pressure, and LAP = mean left atrial pressure.

Arterial blood gas measurements were performed after a 20-min period of ventilation with pure oxygen, before institution of CPB, and 30 and 60 min after reperfusion.

Evaluation of Pulmonary and Lower Limb Skeletal Muscle Leukocyte Sequestration

Blood samples were drawn from the pulmonary artery catheter and left atrial line for total and differential leukocyte counts (Argos 3; ABX France, Montpellier, France) before CPB initiation, at the time of pulmonary reperfusion, and 30 and 60 min after CPB discontinuation.

Myeloperoxidase (MPO) activity, which is an index of PMN tissue sequestration, was determined in lung and lower limb skeletal muscle by the method of Mullane and colleagues (14). MPO activity in skeletal muscle was considered as a test of PMN sequestration in the systemic microvascular bed. Biopsy samples were frozen in liquid nitrogen and stored at 80° C. They were then pulverized and homogenized in 10% wt/vol hexadecyltrimethyl ammonium bromide (HTAB) buffer (0.5% HTAB in 50 mM phosphate buffer at pH 6.0) using a Polytron homogenizer. The homogenate was sonicated on ice for 15 s, frozen at 70° C, and thawed three times, then centrifuged at 40,000 × g for 15 min. Spectrophotometry was used to assay MPO activity in the supernatant. Twenty microliters of supernatant were combined with 12 µl of 25 mM H₂O₂, 30 µl of 40 mM O-dianisidine hydrochloride, and 1.938 ml of 50 mM phosphate buffer (pH 6.0). The change in absorbance was measured at 460 nm on a Beckman spectrometer (Model 25 spectrometer; Beckman, St. Aubin, France). One unit of MPO activity was defined as the activity degrading 1 µmol of peroxide per minute at 25° C.

Isolated Pulmonary and Femoral Arterial Ring Studies

At the end of each experiment, intrapulmonary and femoral arterial segments were dissected out and placed in warm Krebs-Henseleit buffer composed of (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂ · 2 H₂O, 1.2 KH₂PO₄, 1.2 MgSO₄ · 7 H₂O, 25 NaHCO₃, 0.03 EDTA, and 11.1 glucose. Isolated pulmonary and femoral arteries were cleaned and cut into rings 3-4 mm in length (1-2 mm outer diameter). Three to four rings were obtained from each animal. The rings were then mounted on stainless steel hooks, suspended in 10-ml tissue baths, and connected to force displacement transducers (LB-5; Showa-sokki, Japan) for force change recording using a chart recorder (LR 4210; Yokogawa, Japan). The baths were filled with 10 ml of Krebs-Henseleit buffer and aerated at 37° C with a mixture of 95% O₂-5% CO₂. Pulmonary and femoral arterial rings were initially stretched to produce a preload of 1 × g and 3 × g, respectively, and were then allowed to equilibrate for 60-90 min. Pilot studies using length-tension analysis of pulmonary artery rings showed that these preload values provide optimal resting tension. This value is similar to the optimal resting tension (1.060 ± 0.040 × g) found by Liu and coworkers (15) in piglet neonatal pulmonary arterial rings. During this period, the Krebs-Henseleit buffer in the tissue baths was replaced every 10 min. After incubation with indomethacin (10⁵ M) for 60 min, a concentration-response curve to phenylephrine (PE) was obtained. The rings were then washed, and the developed force was allowed to return to baseline. The rings were then precontracted with PE to generate approximately 1 × g of developed force. Once a stable contraction was obtained, cumulative doses of acetylcholine (10⁹ to 10³ M) were added to the bath to assess changes in endothelium-dependent relaxation. The rings were washed again and allowed to equilibrate to baseline. The procedure was repeated with a single dose (10⁵ M) of sodium nitroprusside, an endothelium-independent vasodilator.

Responses were also assessed by determining the concentration that produced 50% of the maximal response (EC₅₀) extrapolated from a plot of log concentration versus percentage of maximal response. The contractile response to phenylephrine was expressed in absolute values (mg), and the maximum relaxation to acetylcholine and sodium nitroprusside as the percentage of the phenylephrine-induced precontraction, with 0% indicating no relaxation and 100% a relaxation of the same magnitude as the precontraction. Femoral arterial ring reactivity was considered as a test for vasoreactivity of the systemic vascular bed.

Cell Preparation

Endothelial cells. Using a previously described technique with a few small modifications (16), porcine pulmonary artery (PAECs) and aortic (AECs) endothelial cells were collected at the end of the experiment by gently scraping the luminal surface of longitudinally opened fresh pulmonary artery (PA) and aorta (Ao) segments. After suspension in culture medium (RPMI 50%, M199 50%) containing 20% heat-inactivated bovine calf serum, the cells were seeded on gelatin-coated 35-mm dishes. Nonadherent cells were removed after 2 h. Confluent endothelial cells were passed after treatment with trypsin-EDTA (0.05% and 0.02%, respectively). Endothelial cell purity was assessed based on detection of the typical cobblestone appearance under the inverted microscope. This method was validated in an earlier study versus von Willebrand factor antigen markers (17). All experiments were performed by seeding second-passage endothelial cells on 24-well gelatin-coated culture plates.

Blood cells. Whole blood was harvested before the operation, 30 and 90 min after CPB initiation, and 30 and 60 min after CPB discontinuation. Whole blood (1 ml), plasma (500 µl), PMNs (4 · 10⁶/ml), monocytes/lymphocytes (4 · 10⁶/ml), and erythrocytes (500 µl) were prepared from various blood groups according to the following methods.

PMNs were purified as described elsewhere (18) using a process involving sedimentation on 2% dextran T-500 and centrifugation on a Ficoll-Paque density gradient, followed by hypotonic lysis of residual erythrocytes. Preparations contained more than 95% PMNs, and viability as assessed by trypan blue exclusion was greater than 97%.

Monocyte/lymphocyte mixtures (M-L) were harvested after Ficoll-Paque sedimentation gradient. The cells were aspirated, resuspended in Hanks' balanced salt solution (HBSS), washed, and counted. Erythrocytes were isolated after whole blood sedimentation on 2% dextran.

PMN Adherence to Endothelial Cells

PMNs (10⁶/ml) were added to washed PAECs obtained from intact animals that did not undergo CPB. These endothelial cells were stimulated with tumor necrosis factor- (TNF-) and calcium ionophore (CaI) for 4 h and 15 min, respectively. Unstimulated control cells were run in parallel. After 30 min of contact, unbound PMNs were removed by three washings with buffer. Adherent PMNs were collected, lysed (1% triton X-100), and sonicated three times for 10 s each time. MPO in the adherent PMN fraction was measured as previously described (17, 18), and adhesion was expressed as the percentage of adherent PMNs relative to the total number of PMNs added at the beginning of the experiment (10⁶ PMNs/well).

Pulmonary Artery and Aortic Endothelial Cell Viability Determination and Cytotoxic Effect of Leukocytes on Pulmonary Artery Endothelial Cells

PAECs and AECs were isolated as described previously, and the percentage of cell necrosis was assessed using crystal violet staining and trypan blue exclusion. AEC viability was taken as an index of systemic endothelium viability.

To assess cytotoxicity of leukocyte on pulmonary artery endothelial cells, whole blood harvested 30 min and 90 min after CPB initiation and 60 min after CPB termination was incubated for 60 min with intact cultured PAECs. Cells were then washed and incubated for 4 h with fresh culture medium. To determine which element was cytotoxic, whole blood was separated into its major constituents (plasma, PMNs, M-L, and erythrocytes), which were again incubated for 60 min with intact cultured PAECs.

Endothelial Cell Proliferation

PAECs and AECs were isolated as described above. The same number of cells (2 · 10⁵ cells/well) were seeded on the wells, and the medium was changed on alternate days until the seventh day. Cells were then harvested (trypsin/EDTA), counted, and seeded for a second 7-d culture period. AEC proliferation was taken as an index of systemic endothelium proliferation.

Reagents and Drugs

The following drugs were used (Sigma Chemical, St. Louis, MO): indomethacin, phenylephrine, acetylcholine, and sodium nitroprusside. All drugs were freshly prepared on the day of experiment.

M199 culture medium, RPMI culture medium, fetal bovine serum, L-glutamine (200 mmol/L), penicillin-streptomycin (5,000 µg/ml- 5,000 UI/ml), fungizone (250 µg/L), trypsin-EDTA (0.05%-0.02%, respectively), and HBSS were from Gibco (Cergy-Pointoise, France); TNF- from Promo Cell (Heidelberg, Germany); and Triton X-100, calcium ionophore, and orthodianisidine from Sigma Chemical. The 24-well gelatin-coated culture plates were obtained from Corning Glass (Corning, NY), and dextran T-500 and Ficoll-Paque from Pharmacia Biotech (Uppsala, Sweden).

Protocol

Eight piglets underwent the entire procedure, i.e., total CPB for 90 min and survival for up to 1 h after CPB. In four other animals, CPB was interrupted before lung reperfusion to individualize the effects of lung ischemia alone versus lung ischemia-reperfusion. In these piglets, all hemodynamic, biochemical, physiologic, and cellular investigations were completed. Six control piglets underwent a sham procedure consisting of median stenotomy, heparin administration in a dose of 2.5 mg/kg, hemodynamic assessment before and after the procedure, and harvesting of pulmonary and femoral arterial rings and pulmonary and aortic endothelial cells. Sham animals did not receive hypothermia and were not exposed to donor blood.

Statistical Analysis

All results are given as means ± SE. Mean values were compared using analysis of variance (ANOVA). The Newman-Keuls multiple-sample comparison test was used to evaluate any differences in results. p Values less than 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Hemodynamic Results

Hemodynamic variables in sham animals were similar before and after the surgical procedure, and were also similar to the pre-CPB values in the two CPB groups (Figure 1). Pulmonary venous return drained to the reservoir during CBP was about 0.5% of the bypass flow.

View larger version (20K): [in this window] [in a new window]   Figure 1.   Hemodynamic results. (A) Systemic vascular resistance index (SVRI) before and after cardiopulmonary bypass (CPB) termination. *p = 0.0001 versus T0; +p = 0.004 versus T0. (B) Pulmonary vascular resistance index (PVRI) before and after CPB termination. *p = 0.0008 versus T0; +p = 0.04 versus T0. (C ) Cardiac index (CI) before and after CPB termination. *p = 0.0001 versus T0; +p = 0.0001 versus T0. (D) PO2 before and after CPB termination. *p = 0.01 versus T0; +p = 0.003 versus T0. T0 = before CPB, T1 = 30 min after CPB termination, T2 = 60 min after CPB termination.

The SVRI values were increased at 30 and 60 min after CPB termination without cross-clamping of the aorta (659.8 ± 104.8 dynes · s · cm⁵ · kg¹ before CPB versus 4,593.2 ± 1,551.4, 30 min after CPB termination [p = 0.0001] and 2,553.8 ± 303.5, 60 min after CPB termination [p = 0.04]).

The PVRI values were increased after weaning from CPB (135.9 ± 374.7 dynes · s · cm⁵ · kg¹ before CPB versus 1,996.7 ± 374.7, 30 min after CPB termination [p = 0.0008] and 1,255.9 ± 458.9, 60 min after CPB termination [p = 0.04]).

The CI was severely diminished after CPB termination (144.0 ± 13.7 ml · min¹ · kg¹ before CPB versus 54.8 ± 6.3, 30 min after CPB termination [p = 0.0001] and 75.6 ± 22.8, 60 min after CPB termination [p = 0.005]).

Pa_O2 was also significantly affected by CPB (Pa_O2 was 508.4 ± 25.7 mm Hg before CPB versus 278.5 ± 81.3, 30 min after CPB termination [p = 0.01] and 221.8 ± 85.7, 60 min after CPB termination [p = 0.003]).

Pulmonary and Peripheral Muscle Leukocyte Sequestration

Pulmonary leukosequestration was indirectly assessed by the difference in pulmonary artery and left atrium leukocyte counts divided by the pulmonary artery leukocyte count and multiplied by 100 (Figure 2). There was a trend for an increase in percentages of PMN sequestration in the lung parenchyma after CPB termination (8.2 ± 7.2% before CPB versus 17.2 ± 37.2% after 30 min CPB initiation and 22.7 ± 24.2%, 60 min after CPB termination; p = NS). Lung MPO activity rose significantly from 0.14 ± 0.01 IU/100 mg of fresh lung tissue before CPB to 0.47 ± 0.04, 60 min after CPB termination; p = 0.001. Intermediate values were not different from baseline (0.16 ± 0.01 after 30 min CPB initiation, 0.20 ± 0.01 after 90 min CPB initiation, and 0.22 ± 0.01, 30 min after CPB termination). There was no increase in MPO activity in skeletal muscle throughout the study.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Lung and skeletal muscle myeloperoxidase activity before, during, and after cardiopulmonary bypass (CPB) termination. *p = 0.001 versus pre-CPB value; +p = 0.0001 versus skeletal muscle myeloperoxidase (MPO) activity.

Isolated Pulmonary and Femoral Arterial Ring Study

The EC₅₀ to phenylephrine was similar for all the pulmonary artery rings obtained from the three experimental groups (Figure 3). The responses to phenylephrine of femoral artery rings were also similar in the three groups (Figure 4).

View larger version (20K): [in this window] [in a new window]   Figure 3.   Endothelium-dependent relaxation of pulmonary artery rings. Open circles are for the rings obtained from pulmonary arteries of sham piglets; closed circles for the rings obtained from pulmonary arteries of piglets undergoing cardiopulmonary bypass (CPB) and lung reperfusion; triangles for the rings obtained from pulmonary arteries of piglets undergoing CPB without lung reperfusion. p = 0.001: Comparison of post-reperfusion versus pre-reperfusion or sham.

View larger version (19K): [in this window] [in a new window]   Figure 4.   Endothelium-dependent relaxation of femoral artery rings. Open circles are for the rings obtained from femoral arteries of non-CPB piglets, and closed circles are for the rings obtained from femoral arteries 60 min after termination of CPB. Maximal response (Emax) and concentration of agonist yielding 50% of maximal response (EC50) were in non-CPB animals: Emax = 119 ± 10% and EC50 = 4.76 · 109 ± 1.70 · 109 M. Sixty minutes after the end of CPB, these values were: Emax = 119 ± 16% and EC50 = 1.66 · 108 ± 7.10 · 109 M (p = NS).

The maximal relaxation response to acetylcholine, expressed as a percentage of the control value in sham animals, was 60% after 90 min CPB and 25% 90 min after CPB termination (p = 0.01). EC₅₀ values for acetylcholine ranged from 8.5 · 10⁸ to 1.8 · 10⁷ M. Conversely, the maximal responses to sodium nitroprusside were not altered after CPB termination, ranging from 100 to 120%. In contrast to the results obtained with pulmonary artery rings, relaxation response to acetylcholine of femoral vessels was not altered after CPB termination.

Endothelial Cell Viability

Percentages of cellular necrosis in endothelial cell samples harvested from pulmonary artery and aorta 60 min after CPB initiation were 5 ± 0.8% and 4.9 ± 0.5%, respectively, i.e., similar to the percentage in the sham group (5.1 ± 0.3%). When endothelial cells were isolated from pulmonary artery and aorta segments after CPB termination, the proportion of necrosis was increased to 55 ± 5% and 51 ± 6% (p = 0.002, as compared to the pre-reperfusion or sham group values).

Endothelial Cell Proliferation

In the sham group and in the group studied at the end of the CPB period, no difference in cell growth was observed between PAECs and AECs at the same time points (7 and 14 d) (Figure 5). These endothelial cell growth patterns were similar to that of the AECs from the group studied 60 min after CPB termination. However, 60 min after CPB termination, PAECs grew faster at both time points (7 and 14 d, p = 0.01) than PAECs harvested before CPB termination or in sham animals, or AECs harvested before or after termination of CPB.

View larger version (12K): [in this window] [in a new window]   Figure 5.   Endothelial cell proliferation. Endothelial cells were harvested from pulmonary artery (PAECs) and aorta (AoECs) segments after reperfusion, counted, and seeded at 2 · 105 cells per well. The medium was changed on alternate days, and the cells were counted on the seventh day. Cells were seeded (2 · 105 cells/ well) and allowed to grow for a further 7-d period. Cells harvested from pulmonary arteries and aortas of sham neonatal piglets not subjected to CPB were run in parallel to serve as controls. *p = 0.01 versus AoECs and sham ECs.

Cytotoxic Effect of Leukocytes on PAECs

As shown in Figure 6, neither plasma nor erythrocytes induced endothelial cellular death, whereas PMN-induced endothelial cell death rates were 22.5% ± 2.1, 30 min after CPB initiation, 31% ± 2.2, 90 min after CPB initiation, and 35.75% ± 3.3, 60 min after CPB termination. Compared with PMNs, monocytes/lymphocytes induced lower (p < 0.05) cell death rates (18.5% ± 1.3, after 30 min CPB initiation, 23.25% ± 2.1 after 90 min CPB initiation, and 28% ± 2.9, 60 min after CPB termination).

View larger version (31K): [in this window] [in a new window]   Figure 6.   Endothelial cytotoxicity induced by circulating blood. Whole blood (WB), erythrocytes (RBC), plasma, polymorphonuclear neutrophils (PMNs), and monocytes/lymphocytes (M/L) were harvested at various times of cardiopulmonary bypass (CPB) (before CPB, 30 min and 90 min after CPB initiation, and 60 min after CPB termination) and incubated for 60 min with intact pulmonary artery endothelial cells (PAECs) harvested from non-CPB animals. At the end of the incubation, the endothelial cells were washed and incubated for 4 h with culture medium only. Cytotoxicity was determined as described in METHODS. Data are means of five experiments, each done in duplicate. WB, PMNs, and M/Ls induced greater endothelial cell cytotoxicity than RBCs or plasma alone. *p < 0.05 versus WB, plasma, and RBC.

Adhesion of PMNs to Endothelial Cells

Adhesion to unstimulated PAECs from intact neonatal piglets of PMNs isolated before CPB (control), then after 30 and 90 min of CPB initiation, was similar to adhesion of fresh PMNs from blood donors (Figure 7). In contrast, adhesion of PMNs isolated 60 min after CPB termination was increased by 25.1 ± 2.74% (p < 0.01) as compared with donor PMN or pre-reperfusion values.

View larger version (17K): [in this window] [in a new window]   Figure 7.   Polymorphonuclear neutrophil (PMN) adhesion to unstimulated and stimulated pulmonary artery endothelial cells (PAECs) harvested from non-CPB animals. PMNs were isolated after various times of CPB: before the operation (control), from blood donors (BD), 30 min and 90 min after CPB initiation, and 60 min after CPB termination. Adherence was determined as described in METHODS. Endothelial cells were stimulated with tumor necrosis factor (TNF) and Cal for 4 h and 30 min, respectively. At the end of the incubation, endothelial cells were washed, 106 PMN/ml were allowed to interact, and adherence was determined as described in METHODS. *p < 0.05 versus all other time points within unstimulated endothelial cell group.

When endothelial cells were stimulated with CaI and TNF, adhesion of PMNs harvested at any time of the experimentation, except at 60 min after termination of CPB, were significantly increased by 37.25 ± 4.2% and 54.5 ± 3.5% (p = 0.002), respectively, as compared with unstimulated endothelial cells at the same experimental time. Although there was an increase in PMN adhesion to unstimulated endothelial cells 60 min after CPB termination, it was still lower than that observed when endothelial cells were stimulated with CaI and TNF (25.12% ± 2.74 versus 37.25 ± 4.2%, p < 0.05) (Figure 7).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our data show that 1 h after the end of total CPB in neonatal piglets, the viability of endothelial cells harvested from pulmonary and systemic arteries was similarly decreased by about 50% as a result of cytotoxic effects of circulating leukocytes. However, severe endothelial dysfunction occurred only in the pulmonary vascular bed and was probably caused by ischemia-reperfusion injury.

The mechanism of the inflammatory response to total CPB includes two components: one is related to the contact of blood with nonphysiologic surfaces during bypass (19), and the other to lung ischemia-reperfusion injury (13). The respective contributions of each of these two components to the inflammatory response to CPB has not yet been defined. Most studies have centered on the expression of adhesive molecules on the surface of leukocytes (20), but have not sought to relate this expression to leukocyte-endothelium interactions or to endothelial injury. We developed an in vitro model to study leukocyte-endothelial cell interactions at various steps of total CPB, and we sought to correlate these interactions to vascular resistance, endothelial function, and PMN sequestration in the systemic and pulmonary vascular beds. Systemic endothelial cell viability and proliferation were assessed in aortic endothelial cells, systemic vascular reactivity in femoral arteries, and systemic leukocyte sequestration in lower limb skeletal muscles.

Although PMNs have been reported to increase the expression of their ligand CD11/CD18 during CPB (21), PMNs obtained 30 and 90 min after CPB initiation, before lung reperfusion, did not adhere to unstimulated endothelial cells. However, when these endothelial cells were stimulated by calcium ionophore, an agonist reported to induce P-selectin and platelet-activating factor expression, or by TNF, which induces expression of intercellular adhesion molecule (ICAM)-1 and E-selectin, PMN adhesion increased markedly relative to unstimulated endothelial cells. This finding suggests that stimulation of endothelial cells is a prerequisite for increased adhesion of PMNs activated by the contact of blood with nonphysiologic surfaces during CPB. By contrast, adhesion to nonstimulated endothelial cells increased when PMNs were harvested 1 h after the termination of CPB, indicating that lung reperfusion injury caused further activation of PMNs, which then became able to induce expression of adhesion molecules by endothelial cells. Pulmonary and aortic endothelial cell viability remained normal during CPB but decreased after reperfusion. During and after CPB, we also observed a time-dependent increase in in vitro PMN- and monocyte-related pulmonary endothelial cell cytotoxicity, which culminated at the end of the reperfusion period, whereas plasma isolated after various CPB durations did not induce pulmonary endothelial cell cytotoxicity. This finding casts doubt on the contribution of soluble factors and demonstrates that CPB-related endothelial cell necrosis is caused primarily by leukocytes. Thus, leukocytes are stimulated during CPB to adhere to stimulated endothelial cells, but ischemia-reperfusion further stimulates leukocyte adherence and cytotoxicity.

Ischemia and reperfusion are increasingly viewed as two separate events, each of which causes specific lung lesions (22). During CPB in our study, the lungs were not ventilated, the pulmonary arterial trunk was clamped to avoid anterograde flow to the lungs, and collateral pulmonary blood was minimal, averaging 0.5% of bypass flow. Although our technique of measurement of collateral pulmonary blood may ignore some part of the bronchial blood flow that returns to the azygous and hemiazygous veins, we believe that the lungs remained ischemic and hypoxic throughout the 90-min CPB period. This was supported by our previous findings of a decrease in lung ATP concentrations during CPB in the same piglet preparation (13). We found a slight decrease in pulmonary endothelium-dependent relaxation at the end of the ischemic period in response to the receptor-mediated endothelium-dependent agonist acetylcholine, whereas maximal responses to the nonendothelium-dependent vasodilator nitroprusside remained normal. This indicates that loss of endothelium-dependent relaxation was not due to an inability of the smooth muscle to relax in response to nitric oxide (NO), but was related to impaired release of NO by the endothelial cells. This result is consistent with previous findings from our group using the isolated rabbit lung preparation (23), and with a recent study from Huk and associates (24) showing a decrease in basal and stimulated endothelium NO release in ischemic skeletal muscle, possibly related to a decrease in L-arginine availability. Ischemia may also initiate an inflammatory response since exposure of endothelial cells to hypoxia leads to induction of interleukin-1 and interleukin-8, followed by increased expression of intercellular adhesion molecules (25, 26). During pulmonary anoxic ischemia, endothelial ATP levels decrease, hypoxanthine levels increase, and xanthine dehydrogenase is converted to the oxidant-generating xanthine oxidase. Reperfusion and reoxygenation is associated with an influx of molecular oxygen that precipitates a burst of oxygen-derived free radicals. This oxidative milieu generates lipid peroxides that activate phospholipase A₂ and trigger the endothelial expression of adhesion molecules and the release of proinflammatory mediators and cytokines (27). Our in vitro findings suggest that these events in turn lead to adhesion of leukocytes to endothelial cells and further increase the CPB-related inflammatory response. Activation of adherent PMNs magnifies endothelial injury by causing production of reactive oxygen species and release of cytotoxic enzymes. In earlier studies of isolated neonatal piglet lungs subjected to ischemia and of intact neonatal piglets subjected to total CPB (13, 28), reperfusion induced severe lung injury characterized by pulmonary edema, pulmonary hypertension, and endothelial dysfunction. In the present study, the deterioration in gas exchange following CPB was indirect evidence of lung injury, and the increase in lung myeloperoxidase, which is a marker of tissue PMN infiltration, indicated that acute pulmonary inflammation occurred. Pulmonary hypertension after reperfusion was associated with a further aggravation in endothelial dysfunction, suggesting a cause-and-effect relationship. The additional decrease in NO activity during reperfusion may therefore contribute to the pathophysiology of reperfusion injury (24).

Surprisingly, although the CPB-related inflammatory process induced similar rates of cellular necrosis among systemic and pulmonary artery endothelial cells, it had opposite consequences on vascular physiology and endothelial function in systemic and pulmonary arteries. First, both at CPB termination and after reperfusion, endothelium-dependent relaxation was selectively decreased in the pulmonary arteries but not in the femoral arteries, suggesting that the endothelial cells in the systemic vascular bed had normal NO activity. Endothelial cell viability was assessed using trypan blue exclusion, which identifies lysed cells but not apoptotic cells (29). Thus, the possibility exists that some of the pulmonary endothelial cells identified as viable were apoptotic. Reactive oxygen species produced in the vicinity of the pulmonary endothelium during reperfusion may have induced endothelial cell apoptosis, and this phenomenon may have been partly responsible for the observed decrease in NO activity in the pulmonary arterial bed (30). Second, after reperfusion, the pulmonary endothelial cells exhibited an increase in their growth rate, whereas the aortic endothelial cells had growth and proliferation patterns similar to the sham pulmonary and aortic endothelial cells. Recent findings that oxidant stress upregulates genes involved in the control of cell proliferation may explain this result (31). Lastly, lung MPO content, a marker of PMN tissue infiltration, was close to pre-CPB values during lung ischemia and increased progressively after reperfusion, whereas peripheral skeletal muscle MPO remained unchanged. This indicates that strong PMN adhesion to endothelial cells and transmigration occurred only in the pulmonary microvascular bed. This agrees with our in vitro observations showing that activated PMNs adhered only after endothelial cell stimulation, an event that probably occurred only in the pulmonary vascular bed as part of the response to the ischemia-reperfusion lung injury.

In conclusion, total CPB in neonatal piglets causes similar rates of endothelial cell death in the systemic and pulmonary vascular beds, as a result of ischemia-reperfusion-induced increases in adherence and cytotoxicity of leukocytes. However, NO activity of the remaining viable endothelial cells was depressed in the pulmonary vascular bed only in association with PMN sequestration in the lung but not in the systemic microvascular bed. These findings provide evidence that lung ischemia-reperfusion aggravates the inflammatory response to CPB and predispose the lung endothelium to leukocyte-mediated injury.