Eugenio G. Araujo

Gene expression profile after cardiopulmonary bypass and cardioplegic arrest

OBJECTIVE This study examines the cardiac and peripheral gene expression responses to cardiopulmonary bypass and cardioplegic arrest. METHODS Atrial myocardium and skeletal muscle were harvested from 16 patients who underwent coronary artery bypass grafting before and after cardiopulmonary bypass and cardioplegic arrest. Ten sample pairs were selected for patient similarity, and oligonucleotide microarray analyses of 12,625 genes were performed using matched precardiopulmonary bypass tissues as controls. Array results were validated with Northern blotting, real-time polymerase chain reaction, in situ hybridization, and immunoblotting. Statistical analyses were nonparametric. RESULTS Median durations of cardiopulmonary bypass and cardioplegic arrest were 74 and 60 minutes, respectively. Compared with precardiopulmonary bypass, postcardiopulmonary bypass myocardial tissues revealed 480 up-regulated and 626 down-regulated genes with a threshold P value of.025 or less (signal-to-noise ratio: 3.46); skeletal muscle tissues showed 560 and 348 such genes, respectively (signal-to-noise ratio: 3.04). Up-regulated genes in cardiac tissues included inflammatory and transcription activators FOS; jun B proto-oncogene; nuclear receptor subfamily 4, group A, member 3; MYC; transcription factor-8; endothelial leukocyte adhesion molecule-1; and cysteine-rich 61; apoptotic genes nuclear receptor subfamily 4, group A, member 1 and cyclin-dependent kinase inhibitor 1A; and stress genes dual-specificity phosphatase-1, dual-specificity phosphatase-5, and B-cell translocation gene 2. Up-regulated skeletal muscle genes included interleukin 6; interleukin 8; tumor necrosis factor receptor superfamily, member 11B; nuclear receptor subfamily 4, group A, member 3; transcription factor-8; interleukin 13; jun B proto-oncogene; interleukin 1B; glycoprotein Ib, platelet, alpha polypeptide; and Ras-associated protein RAB27A. Down-regulated genes included haptoglobin and numerous immunoglobulins in the heart, and factor H-related gene 2, protein phosphatase 1, regulatory subunit 3A, and growth differentiation factor-8 in skeletal muscle. CONCLUSIONS By establishing a profile of the gene-expression responses to cardiopulmonary bypass and cardioplegia, this study allows a better understanding of their effects and provides a framework for the evaluation of new cardiac surgical modalities directly at the genome level.

Aprotinin Preserves Cellular Junctions and Reduces Myocardial Edema After Regional Ischemia and Cardioplegic Arrest

Background—Cardiac surgery with cardiopulmonary bypass (CPB) and cardioplegic arrest has been associated with myocardial edema attributable to vascular permeability, which is regulated in part by thrombin-induced alterations in cellular junctions. Aprotinin has been demonstrated to prevent activation of the thrombin protease-activated receptor, and we hypothesized that aprotinin preserves myocardial cellular junctions and prevents myocardial edema in a porcine model of regional ischemia and cardioplegic arrest. Methods and Results—Fourteen pigs were subjected to 30 minutes of regional ischemia, followed by 60 minutes of CPB, with 45 minutes of crystalloid cardioplegia, then 90 minutes of post-CPB reperfusion. The treatment group (n=7) was administered aprotinin (40 000 kallikrein inhibitor units [KIU]/kg loading dose, 40 000 KIU/kg pump prime, and 10 000 KIU/kg per hour continuous infusion). Control animals (n=7) received normal saline. Myocardial vascular endothelial (VE)-cadherin, &bgr;-catenin and &ggr;-catenin, and associated mitogen-activated protein kinase (MAPK) pathways were assessed by immunoblot and immunoprecipitation. Histologic analysis of the cellular junctions was done by immunofluorescence. Myocardial tissue water content was measured. VE-cadherin, &bgr;-catenin, and &ggr;-catenin levels were significantly greater in the aprotinin group (all P<0.05). Immunfluorescence confirmed that aprotinin prevented loss of coronary endothelial adherens junction continuity. Aprotinin reduced tyrosine phosphorylation in myocardial tissue sections. Phospho-p38 activity was ≈30% lower in the aprotinin group (P=0.007). The aprotinin group demonstrated decreased myocardial tissue water content (81.2±0.5% versus 83.5±0.3%; P=0.01) and reduced intravenous fluid requirements (2.9±0.2 L versus 4.0±0.4 L; P=0.03). Conclusions—Aprotinin preserves adherens junctions after regional ischemia and cardioplegic arrest through a mechanism potentially involving the p38 MAPK pathway, resulting in preservation of the VE barrier and reduced myocardial tissue edema.

Activation of pulmonary mitogen-activated protein kinases during cardiopulmonary bypass.

PURPOSE Cardiopulmonary bypass (CPB) produces an inflammatory response associated with pulmonary dysfunction. Mitogen-activated protein kinases (MAPK) have been shown to mediate pulmonary injury. We hypothesized that MAPK are activated during CPB and potentially contribute to lung injury. METHODS Pigs were placed on CPB (n = 6) for 90 min, which included 80 min of cardioplegic arrest, followed by 180 min of post-CPB reperfusion. Control animals (n = 6) underwent sternotomy and heparinization only. Lung samples were collected at baseline, during CPB, and during post-CPB reperfusion. Activated forms of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 were measured by Western blot. Immunohistochemistry was used for tissue localization of activated MAPK. Pulmonary inflammation was determined by histology. Pulmonary edema was estimated by tissue water percentage. RESULTS Activated ERK1/2 and p38 were increased after 90 min of CPB compared with controls (3.94 +/- 0.61- and 2.49 +/- 0.15-fold increase, respectively; both P < 0.01). At 180 min of post-CPB reperfusion, ERK1/2 activity was increased by nearly 5-fold compared with controls (P < 0.01), whereas p38 activity returned to baseline levels. By immunohistochemistry, activated ERK1/2 and p38 in the CPB group were localized to alveolar epithelial cells, vascular endothelial cells, and bronchial smooth muscle. Histologic signs of lung injury included leukocyte infiltration in the CPB group. Tissue water percentage was increased with CPB (89.9 +/- 1.5% versus 82.5 +/- 1.0%, CPB versus control, P < 0.05). CONCLUSIONS The results of our study demonstrate that CPB increases pulmonary p38 activity and causes sustained activation of ERK1/2. MAPK activation thus may in part mediate the pulmonary inflammatory response and provide a potential site of intervention to prevent pulmonary dysfunction due to CPB.