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Induction of Higher Expression of IL-1β and TNF-α, Lower Expression of IL-10 and Cyclic Guanosine Monophosphate by Pulmonary Arterial Hypertension Following Cardiopulmonary Bypass
Asian Journal of Surgery ©
Excerpta Medica Asia Ltd
β and TNF-α α, Induction of Higher Expression of IL-1β Lower Expression of IL-10 and Cyclic Guanosine Monophosphate by Pulmonary Arterial Hypertension Following Cardiopulmonary Bypass Ye Lei,1 Jiang Zhen,2 Xia L. Ming3 and Hu K. Jian,3 1Clinical Research Center, Department of Surgery, National University of Singapore, Singapore, 2Department of Anaesthesiology, 3Department of Cardiac Surgery, Zhongshan Hospital, Shanghai Cardiovascular Diseases Institute, Medical Center of Fudan University, Shanghai, People’s Republic of China. OBJECTIVE: Pulmonary arterial hypertension (PAH) and cardiopulmonary bypass (CPB) induce systemic inflammatory cytokines that are critical factors related to postoperative mortality of open heart surgery. We studied the expression of proinflammatory cytokines and cyclic guanosine monophosphate (cGMP) in patients suffering from PAH after CPB. METHODS: Seventy-six patients who underwent valve replacement surgery were recruited and divided into two groups according to their pulmonary arterial pressure (< 50 mmHg for Group A and ≥ 50 mmHg for Group B). Blood samples were taken to measure the concentrations of interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10) and cGMP. RESULTS: IL-1β and TNF-α were significantly higher in Group B (28.6 ± 9.1 mmHg) than in Group A (65.8 ± 10.2 mmHg) at baseline. After CPB, IL-1β of both groups rose significantly, while only TNF-α of Group B rose significantly higher. There were significant differences between the two groups after CPB. IL-10 and cGMP in Group B were lower than in Group A at baseline. They all decreased significantly after CPB. Significant differences were seen between the groups after CPB. CONCLUSION: Patients suffering from PAH had different levels of proinflammatory and antiinflammatory cytokines compared to normal patients. PAH aggravates the production of IL-1β and TNF-α, while it decreases the production of IL-10 and cGMP after CPB. (Asian J Surg 2002;25(3):203–8)
INTRODUCTION Cardiopulmonary bypass (CPB) is characterized by systemic endotoxaemia followed by cytokine generation, including the proinflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and antiinflammatory cytokine interleukin-10 (IL-10).1,2 The
Address reprint requests to Dr. Ye Lei, Room B1–12, MD11, Clinical Research Center, Department of Surgery, National University of Singapore, Singapore. Email: [email protected] Date of acceptance: 7th March 2002
Asian Journal of Surgery
proinflammatory cytokines TNF-α and IL-1β upregulate neutrophil and endothelial adhesive molecule expression and enhance neutrophil-endothelial adherence. 3–5 Enhanced neutrophil-endothelial adherence is thought to be responsible for inducing lung and myocardial reperfusion injury after CPB.6 The elevated systemic level of these proinflammatory cytokines correlate with increased morbidity and mortality after CPB.1,2,6 Nitric oxide (NO) is the main factor that regulates vasomotion, and its level is closely related to the occurrence of pulmonary arterial hypertension (PAH),7,8 and can be expressed by directly activating cyclic guanosine monophosphate (cGMP).9,10 It has been suggested that proinflammatory cytokines can increase NO via the expression of inducible NO synthase (iNOS). 11,12
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Furthermore, NO is increased after CPB. Therefore, it would be helpful to investigate the effects of PAH on CPB-induced proinflammatory cytokines (IL-1β, TNF-α, and IL-10) and cGMP (as an indicator of NO).
were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s procedure. The reagents for cGMP were provided by Sigma Company (St Louis, MO, USA), and cGMP was assayed using a 125IcGMP radio-immunoassay method.13
PATIENTS AND METHODS STATISTICAL ANALYSIS Seventy-six patients with heart function classified as New York Heart Association class III who underwent mitral and/or aortic valve replacement were studied. The protocol was approved by the Zhongshan Hospital (Shanghai, China) institutional review board and informed consent was obtained from patients. Pulmonary arterial pressure (PAP) was measured by echocardiography before surgery for each patient. Based on the PAP, the patients were divided into two groups (cut-off level = 50 mmHg): Group A with PAP < 50 mmHg (mean = 28.6 ± 9.1 mmHg) and Group B with PAP ≥ 50 mmHg (mean = 65.8 ± 10.2 mmHg) (Table). Surgical procedures were performed as routine general anaesthesia and CPB. Centrifugal pumps and membrane oxygenators were used for all patients. CPB was performed with moderate hypothermia (27°–29°C) and moderate blood dilution (hematocrit: 23%–27%). The activated clotting time was maintained at greater than 480 seconds. Blood flow rate ranged from 50–70 ml/kg. Cardioplegic cold blood (4:1) was administered into the root of aorta or into the left and right sinuses of the coronary arteries. Blood samples were taken via the radial artery before surgery (as baseline), at weaning from CPB and 24 hours after CPB to measure the concentrations of IL-1β, TNF-α, IL-10 and cGMP. The blood samples (collected in 20-ml tubes) were immediately taken to the laboratory and centrifuged at 4,000 rpm for 20 minutes. The plasma was then separated and frozen at –70°C for future use. The reagents for IL-1β, TNF-α and IL-10 were provided by DIACLONE (France). The levels of IL-1β, TNF-α and IL-10
Table.
Group A Group B
All data are given as mean plus or minus standard error of the mean (SEM). The level of statistical significance (p < 0.05) was tested using Student’s t test on paired data.
RESULTS Clinical variables All patients selected suffered from rheumatic heart disease and underwent mitral and/or aortic valve replacement. The demographic and perioperative data are listed in the Table. Except for PAP, there were no significant differences between the two groups related to age, weight, ejection fraction, cross-clamping time or CPB.
Perioperative changes in IL-1β The IL-1β level of Group B (16.8 pg/ml) was higher than that of Group A (10.2 pg/ml) at baseline (p < 0.05). Though the IL-1β level of both groups increased significantly after CPB (p < 0.01), it rose significantly higher in Group B (34.8 pg/ml) than in Group A (25.7 pg/ml) (p < 0.05) (Figure 1).
Perioperative changes in TNF-α The TNF-α level of Group A rose slightly after CPB (16 pg/ml to 19.7 pg/ml), while the TNF-α level of Group
Demographic and perioperative data Cases
Sex (M/F)
Age (y)
Weight (kg)
46 30
30/16 15/15
47.5 ± 11.4 43.5 ± 10.8
57.2 ± 9.5 56.7 ± 9.4
EF (%) 55.5 ± 8.4 49.4 ± 7.8
T-CC (min)
T-CPB (min)
65.8 ± 18.2 64.0 ± 18.6
120.2 ± 30.4 115.8 ± 26.4
M/F = male/ female; EF = ejection fraction; T-CC = time of cross-clamping; T-CPB = time of cardiopulmonary bypass.
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IL-1β (pg/ml)
40
**# **#
** ** 20
Group A
#
Group B
0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: ** p < 0.01 Comparison vs Group A: #p < 0.05
Figure 1.
Change in IL-1β (pg/ml). CPB = cardiopulmonary bypass.
B was higher at baseline, and much higher after CPB (25.3 pg/ml to 133.6 pg/ml) (p < 0.01). The TNF-α differences between the two groups during the perioperative period were significant (p < 0.01) (Figure 2).
10 levels of both groups decreased after CPB (p < 0.05) and tended to recover within 24 hours after CPB. The IL10 levels of the two groups after CPB were significantly different (p < 0.05) (Figure 3).
Perioperative changes in IL-10
Perioperative changes in cGMP
The IL-10 level of Group B (6.2 U/L) was lower than that of Group A (10.3 U/L) at baseline (p < 0.05). The IL-
The cGMP level of Group B (4.1 pmol/ml) was lower than that of Group A at baseline (6.1 pmol/ml) (p < 0.01).
TNF-α (pg/ml)
160
**##
120
**##
80
Group A Group B
##
40 0
Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: ** p < 0.01 Comparison vs Group A: ## p < 0.01 Figure 2.
Change in TNF-α (pg/ml). CPB = cardiopulmonary bypass.
Asian Journal of Surgery
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IL-10 (U/L)
12 8
#
*
4
#
*#
Group A Group B
0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: * p < 0.05 Comparison vs Group A: # p < 0.05
Figure 3.
Change in IL-10 (U/L). CPB = cardiopulmonary bypass.
Though the cGMP levels of both groups decreased significantly after CPB (p < 0.01), they returned to baseline in Group A (p < 0.05) after 24 hours. The cGMP levels of the two groups after CPB were significantly different (p < 0.01) (Figure 4).
DISCUSSION PAH is an important factor for morbidity and mortality during the perioperative period of cardiac surgery,14 and it is suggested that up to 58% of early postoperative
mortality is associated with PAH.15 CPB can aggravate PAH by the formation of emboli, along with increasing thromboxane and induction of the inflammatory response by activating complement and leukocytes, and expressing adherent cytokines.16,17 More attention has been drawn to study the inflammatory response during CPB.1–3,17 CPB is responsible for a systemic inflammatory response, which influences the patient’s recovery after surgery, especially in infants and neonates.18 The cytokines released during CPB include IL-1β, TNF-α and IL-10. IL-1β and TNF-α can accelerate the
cGMP (pmol/ml)
8
4
##
** *## Group A
**##
Group B 0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: * p < 0.05, ** p < 0.01 Comparison vs Group A: ## p < 0.01
Figure 4.
206
Change in cGMP (pmol/ml). CPB = cardiopulmonary bypass.
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EXPRESSION OF IL-Iβ, TNF-α, IL-10 AND cGMP AFTER CPB
inflammatory response, while IL-10 plays a protective role by suppressing inflammatory cytokines.19–21 The mechanism of lung failure after cardiac surgery remains to be defined. Understanding of the factors that control pulmonary vasomotion and the role of NO have increased greatly. NO is the main factor that regulates pulmonary vascular tone.8,9 Its concentration is closely related to the occurrence of PAH. Due to the fact that it is difficult to measure the NO concentration in the body, we measured the cGMP concentration. cGMP level is closely related to NO level.10,11 At baseline, the IL-1β and TNF-α levels in Group A were lower than those in Group B, while the IL-10 level in Group A was higher than that in Group B. This suggests that the balance between proinflammatory and antiinflammatory cytokines at baseline is different among patients with different PAPs. At baseline, patients with PAH may suffer from increased production of IL-1β, TNF-α and decreased production of IL-10. These baseline differences may be the result of abnormally functioning pulmonary arterial endothelium in patients with PAH. Patients with PAH have decreased expression of NOS, which results in decreased release of NO.22 Decreased NO may contribute to the increased levels of proinflammatory cytokines. The increased proinflammatory cytokines also may be related to the pathology of patients undergoing valve replacement surgery and may be responsible for the lower ejection fraction in Group B. We suggest that the balance between proinflammatory response and anti-inflammatory response, which may be important for the clinical outcome, could be different in patients with PAH. The cytokines IL-1β, TNF-α and IL-10 of both groups rose or decreased significantly after CPB (except for TNF-α of Group A). These changes are related to CPB.19–21 The interaction between blood and synthetic surfaces during CPB results in the activation of complement and the coagulation system, neutrophils and leukocytes16,17, 23–25 and induces systemic proinflammatory cytokines2,19–21,26 IL-1β and TNF-α of Group B rose significantly compared with group A after CPB. We believe that IL-1β and TNF-α are more highly expressed after induction in patients with PAH after CPB. Increased TNF-α may contribute to myocardial dysfunction and haemodynamic instability following CPB.27,28 The IL-10 level of Group A remained higher than that of Group B after CPB. We believe that the aggravated inflammatory reaction consumes more IL-10 in patients with PAH. The balance of inflammatory and
Asian Journal of Surgery
anti-inflammatory reactions is upset and is liable to aggravate the inflammatory reaction. The increased levels of IL-1β and TNF-α result in overexpression of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1, 3–5 which promote leukocyte adhesion, filtration and transmigration, leading to vascular damage.29,30 The level of cGMP was significantly lower in Group B at baseline which implies that the endogenous NO, produced by constitutive NOS, is lower in patients with PAH. cGMP was much lower in Group B after CPB compared to Group A. This may show that NO is produced less after CPB in patients with PAH. Research suggests that NO production significantly increases during and after CPB and is induced by proinflammatory cytokines via the expression of iNOS,12,31 especially in the lungs and airways.32,33 The decrease in NO found in our study may have contributed to the decreased expression of NOS as a consequence of PAH.22 Based on our study, it appears that the baseline level of proinflammatory cytokines IL-1β and TNF-α are higher and the anti-inflammatory cytokine IL-10 is lower in patients with PAH. The baseline level of cGMP was also lower in this group of patients. The production of systemic proinflammatory cytokines IL-1β and TNF-α was highly expressed and the anti-inflammatory cytokine IL-10 was less expressed in patients with PAH compared to patients with normal PAP after CPB. Decreased production of cGMP was also observed in this group of patients. These circumstances may be responsible for postoperative organ dysfunction and morbidity and mortality, especially in patients with PAH. Anti-inflammatory therapy may be a potential method to treat PAH during the perioperative period of cardiac surgery.
ACKNOWLEDGEMENT We thank Dr. Xu X. Hui for his expert laboratory work and Dr. J. M. Luk, of the Department of Surgery of the University of Hong Kong for his critical comments.
REFERENCES 1. Prondzinsky R, Muller-Werdan U, Pilz G, et al. Systemic inflammatory reactions to extracorporeal therapy measures (II): cardiopulmonary bypass. Wien Klin Wochenschr 1997; 109:346–53. 2. Hill GE. Cardiopulmonary bypass-induced inflammation: is
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LEI AND OTHERS it important? J Cardiothorac Vasc Anesth 1998;12:21–5. 3. Osborn L, Hwaaion R, Tizard C. Direct cloning of vascular cell adhesion molecule-1 (VCAM-1), a cytokine-induced endothelial protein that binds to lymphocytes. Cell 1989;59: 1203–11. 4. Hattori Y, Kasai K. Induction of mRNAs for ICAM-1, VCAM-1, and ELAM-1 in cultured rat cardiac myocytes and myocardium in vivo. Biochem Mol Biol Int 1997;41: 979–86. 5. Kacimi R, Karliner JS, Koudssi F, Long CS. Expression and regulation of adhesion molecules in cardiac cells by cytokines, response to acute hypoxia. Circ Res 1998;82:576–86. 6. Hill GE, Whitten CW, Landers DF. The influence of cardiopulmonary bypass on cytokines and cell-cell communication. J Cardiothorac Vasc Anesth 1997;11:367–75. 7. Stamler JS, Loh E, Roddy MA, et al. Nitric oxide regular basal systemic and pulmonary vascular resistance in healthy humans. Circulation 1994;89:2035–40. 8. Cooper CJ, Landzberg MJ, Anderson TJ, et al. Role of nitric oxide in the local regulation of pulmonary vascular resistance in humans. Circulation 1996;93:266–71. 9. Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ. Activation of purified soluble guanylate cyclase by endothelium derived relaxing factor from intrapulmonary artery and veins: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Therap 1986;237:893–900. 10. Kondo K, Mitchell JA, Nucci G, Vane JR. Simultaneous measurement of endothelium-derived relaxing factor by bioassay and guanylate cyclase stimulation. Br J Pharmacol 1989;98:630–6. 11. Tsujino M, Hirata Y, Imai T, et al. Induction of nitric oxide synthase gene by interleukin-1b in cultured rat cardiocytes. Circulation 1994;90:375–83. 12. Ungureanu-Longrois D, Balligand JL, Simmons WW, et al. Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to b-adrenergic agonists. Circ Res 1995;77:494–502. 13. Axelsson KL, Bornefeldt KE, Norlander B, Wikberg JE. Attomole sensitive radioimmunoassay for cyclic GMP. Second Messengers Phosphoproteins 1988;12:145–54. 14. Wheller J, George B, Mulder DG, Jarmakani JM. Diagnosis and management of postoperative pulmonary hypertensive crisis. Circulation 1979;60:1640–9. 15. Bando K, Turrentine MW, Sharp TG, et al. Pulmonary hypertension after operations for congenital heart disease: analysis of risk factors and management. J Thorac Cardiovasc Surg 1996;112:1600–9. 16. Koul B, Wollmer P, Willen H, et al. Venoarterial extracorporeal membrane oxygenation — how safe is it? J Thorac Cardiovasc Surg 1992;104:579–84.
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17. Boldt J, Kumle B, Papsdorf M, Hempelmann G. Are circulating adhesion molecules specially changed in cardiac surgical patients? Ann Thorac Surg 1998;65:608–14. 18. Wan S, Leclerc JL, Vincent JL. Inflammatory response to cardiopulmonary bypass. Mechanisms involved and possible therapeutic strategies. Chest 1997;112:676–92. 19. Kristiansson M, Soop M, Saraste L, Sundqvist KG. Cytokines in stored blood cell concentrates: promoters of systemic inflammatory and stimulators of acute transfusion reaction. Acta Anaesthesiol Scand 1996;40:496–501. 20. Drazan KE, Wu L, Bullington D, Shaked A. Viral IL-10 gene therapy inhibits TNF-a and IL-1b, not IL-6, in the newborn endotoxemic mouse. J Pediatr Surg 1996;31:411–4. 21. Chaly YV, Paleolog EM, Kolesnikova TS, et al. Neutrophil adefensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells. Eur Cytokine Netw 2000;11: 257–66. 22. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lung of patients with pulmonary hypertension. N Engl J Med 1995;333:214–21. 23. Gillinov AM, Redmond JM, Zehr KJ, et al. Complement inhibition with soluble complement receptor type 1 in cardiopulmonary bypass. Ann Thorac Surg 1993;55: 619–24. 24. Korthuis RJ, Anderson DC, Granger DN. Role of neutrophilendothelial cell adhesion in inflammatory disorders. J Crit Care 1994;9:47–71. 25. Elliott MJ, Finn AH. Interaction between neutrophils and endothelium. Ann Thorac Surg 1993;56:1503–8. 26. Edmunds LH. Inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1998;66:S12–16. 27. Jansen DG, Velthuis H, Straaten HMO, et al. Perfusionrelated factors of endotoxin release during CPB. Eur J Cardiothorac Surg 1994;8:125–9. 28. Ohkawa F, Ikeda U, Kanbe T, et al. Effects of inflammatory cytokines on vascular tone. Cardiovasc Res 1995;30:711–5. 29. Palluy O, Morliere L, Gris JC, et al. Hypoxia/reoxygenation stimulates endothelium to promote neutrophil adhesion. Free Radic Biol Med 1992;13:21–30. 30. Yoshida N, Granger DN, Anderson DC, et al. Anoxia/ reoxygenation-induced neutrophil adherence to cultured endothelial cells. Am J Physiol 1992;262:H1891–8. 31. Ruvolo G, Greco E, Speziale G, et al. Nitric oxide formation during CPB. Ann Thorac Surg 1994;57:1055–7. 32. Delgado R, Rojas A, Glaria LA, et al. Ca2+-independent nitric oxide synthase activity in human lung after CPB. Thorax 1995;50:403–4. 33. Hill GE, Snider S, Galbraith TA, et al. Glucocorticoid reduction of bronchial epithelial inflammation during CPB. Am J Respir Crit Care Med 1995;152:1791–5.
Vol 25 • No 3 • July 2002
Excerpta Medica Asia Ltd
β and TNF-α α, Induction of Higher Expression of IL-1β Lower Expression of IL-10 and Cyclic Guanosine Monophosphate by Pulmonary Arterial Hypertension Following Cardiopulmonary Bypass Ye Lei,1 Jiang Zhen,2 Xia L. Ming3 and Hu K. Jian,3 1Clinical Research Center, Department of Surgery, National University of Singapore, Singapore, 2Department of Anaesthesiology, 3Department of Cardiac Surgery, Zhongshan Hospital, Shanghai Cardiovascular Diseases Institute, Medical Center of Fudan University, Shanghai, People’s Republic of China. OBJECTIVE: Pulmonary arterial hypertension (PAH) and cardiopulmonary bypass (CPB) induce systemic inflammatory cytokines that are critical factors related to postoperative mortality of open heart surgery. We studied the expression of proinflammatory cytokines and cyclic guanosine monophosphate (cGMP) in patients suffering from PAH after CPB. METHODS: Seventy-six patients who underwent valve replacement surgery were recruited and divided into two groups according to their pulmonary arterial pressure (< 50 mmHg for Group A and ≥ 50 mmHg for Group B). Blood samples were taken to measure the concentrations of interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), interleukin-10 (IL-10) and cGMP. RESULTS: IL-1β and TNF-α were significantly higher in Group B (28.6 ± 9.1 mmHg) than in Group A (65.8 ± 10.2 mmHg) at baseline. After CPB, IL-1β of both groups rose significantly, while only TNF-α of Group B rose significantly higher. There were significant differences between the two groups after CPB. IL-10 and cGMP in Group B were lower than in Group A at baseline. They all decreased significantly after CPB. Significant differences were seen between the groups after CPB. CONCLUSION: Patients suffering from PAH had different levels of proinflammatory and antiinflammatory cytokines compared to normal patients. PAH aggravates the production of IL-1β and TNF-α, while it decreases the production of IL-10 and cGMP after CPB. (Asian J Surg 2002;25(3):203–8)
INTRODUCTION Cardiopulmonary bypass (CPB) is characterized by systemic endotoxaemia followed by cytokine generation, including the proinflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and antiinflammatory cytokine interleukin-10 (IL-10).1,2 The
Address reprint requests to Dr. Ye Lei, Room B1–12, MD11, Clinical Research Center, Department of Surgery, National University of Singapore, Singapore. Email: [email protected] Date of acceptance: 7th March 2002
Asian Journal of Surgery
proinflammatory cytokines TNF-α and IL-1β upregulate neutrophil and endothelial adhesive molecule expression and enhance neutrophil-endothelial adherence. 3–5 Enhanced neutrophil-endothelial adherence is thought to be responsible for inducing lung and myocardial reperfusion injury after CPB.6 The elevated systemic level of these proinflammatory cytokines correlate with increased morbidity and mortality after CPB.1,2,6 Nitric oxide (NO) is the main factor that regulates vasomotion, and its level is closely related to the occurrence of pulmonary arterial hypertension (PAH),7,8 and can be expressed by directly activating cyclic guanosine monophosphate (cGMP).9,10 It has been suggested that proinflammatory cytokines can increase NO via the expression of inducible NO synthase (iNOS). 11,12
203
LEI AND OTHERS
Furthermore, NO is increased after CPB. Therefore, it would be helpful to investigate the effects of PAH on CPB-induced proinflammatory cytokines (IL-1β, TNF-α, and IL-10) and cGMP (as an indicator of NO).
were determined by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s procedure. The reagents for cGMP were provided by Sigma Company (St Louis, MO, USA), and cGMP was assayed using a 125IcGMP radio-immunoassay method.13
PATIENTS AND METHODS STATISTICAL ANALYSIS Seventy-six patients with heart function classified as New York Heart Association class III who underwent mitral and/or aortic valve replacement were studied. The protocol was approved by the Zhongshan Hospital (Shanghai, China) institutional review board and informed consent was obtained from patients. Pulmonary arterial pressure (PAP) was measured by echocardiography before surgery for each patient. Based on the PAP, the patients were divided into two groups (cut-off level = 50 mmHg): Group A with PAP < 50 mmHg (mean = 28.6 ± 9.1 mmHg) and Group B with PAP ≥ 50 mmHg (mean = 65.8 ± 10.2 mmHg) (Table). Surgical procedures were performed as routine general anaesthesia and CPB. Centrifugal pumps and membrane oxygenators were used for all patients. CPB was performed with moderate hypothermia (27°–29°C) and moderate blood dilution (hematocrit: 23%–27%). The activated clotting time was maintained at greater than 480 seconds. Blood flow rate ranged from 50–70 ml/kg. Cardioplegic cold blood (4:1) was administered into the root of aorta or into the left and right sinuses of the coronary arteries. Blood samples were taken via the radial artery before surgery (as baseline), at weaning from CPB and 24 hours after CPB to measure the concentrations of IL-1β, TNF-α, IL-10 and cGMP. The blood samples (collected in 20-ml tubes) were immediately taken to the laboratory and centrifuged at 4,000 rpm for 20 minutes. The plasma was then separated and frozen at –70°C for future use. The reagents for IL-1β, TNF-α and IL-10 were provided by DIACLONE (France). The levels of IL-1β, TNF-α and IL-10
Table.
Group A Group B
All data are given as mean plus or minus standard error of the mean (SEM). The level of statistical significance (p < 0.05) was tested using Student’s t test on paired data.
RESULTS Clinical variables All patients selected suffered from rheumatic heart disease and underwent mitral and/or aortic valve replacement. The demographic and perioperative data are listed in the Table. Except for PAP, there were no significant differences between the two groups related to age, weight, ejection fraction, cross-clamping time or CPB.
Perioperative changes in IL-1β The IL-1β level of Group B (16.8 pg/ml) was higher than that of Group A (10.2 pg/ml) at baseline (p < 0.05). Though the IL-1β level of both groups increased significantly after CPB (p < 0.01), it rose significantly higher in Group B (34.8 pg/ml) than in Group A (25.7 pg/ml) (p < 0.05) (Figure 1).
Perioperative changes in TNF-α The TNF-α level of Group A rose slightly after CPB (16 pg/ml to 19.7 pg/ml), while the TNF-α level of Group
Demographic and perioperative data Cases
Sex (M/F)
Age (y)
Weight (kg)
46 30
30/16 15/15
47.5 ± 11.4 43.5 ± 10.8
57.2 ± 9.5 56.7 ± 9.4
EF (%) 55.5 ± 8.4 49.4 ± 7.8
T-CC (min)
T-CPB (min)
65.8 ± 18.2 64.0 ± 18.6
120.2 ± 30.4 115.8 ± 26.4
M/F = male/ female; EF = ejection fraction; T-CC = time of cross-clamping; T-CPB = time of cardiopulmonary bypass.
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EXPRESSION OF IL-Iβ, TNF-α, IL-10 AND cGMP AFTER CPB
IL-1β (pg/ml)
40
**# **#
** ** 20
Group A
#
Group B
0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: ** p < 0.01 Comparison vs Group A: #p < 0.05
Figure 1.
Change in IL-1β (pg/ml). CPB = cardiopulmonary bypass.
B was higher at baseline, and much higher after CPB (25.3 pg/ml to 133.6 pg/ml) (p < 0.01). The TNF-α differences between the two groups during the perioperative period were significant (p < 0.01) (Figure 2).
10 levels of both groups decreased after CPB (p < 0.05) and tended to recover within 24 hours after CPB. The IL10 levels of the two groups after CPB were significantly different (p < 0.05) (Figure 3).
Perioperative changes in IL-10
Perioperative changes in cGMP
The IL-10 level of Group B (6.2 U/L) was lower than that of Group A (10.3 U/L) at baseline (p < 0.05). The IL-
The cGMP level of Group B (4.1 pmol/ml) was lower than that of Group A at baseline (6.1 pmol/ml) (p < 0.01).
TNF-α (pg/ml)
160
**##
120
**##
80
Group A Group B
##
40 0
Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: ** p < 0.01 Comparison vs Group A: ## p < 0.01 Figure 2.
Change in TNF-α (pg/ml). CPB = cardiopulmonary bypass.
Asian Journal of Surgery
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IL-10 (U/L)
12 8
#
*
4
#
*#
Group A Group B
0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: * p < 0.05 Comparison vs Group A: # p < 0.05
Figure 3.
Change in IL-10 (U/L). CPB = cardiopulmonary bypass.
Though the cGMP levels of both groups decreased significantly after CPB (p < 0.01), they returned to baseline in Group A (p < 0.05) after 24 hours. The cGMP levels of the two groups after CPB were significantly different (p < 0.01) (Figure 4).
DISCUSSION PAH is an important factor for morbidity and mortality during the perioperative period of cardiac surgery,14 and it is suggested that up to 58% of early postoperative
mortality is associated with PAH.15 CPB can aggravate PAH by the formation of emboli, along with increasing thromboxane and induction of the inflammatory response by activating complement and leukocytes, and expressing adherent cytokines.16,17 More attention has been drawn to study the inflammatory response during CPB.1–3,17 CPB is responsible for a systemic inflammatory response, which influences the patient’s recovery after surgery, especially in infants and neonates.18 The cytokines released during CPB include IL-1β, TNF-α and IL-10. IL-1β and TNF-α can accelerate the
cGMP (pmol/ml)
8
4
##
** *## Group A
**##
Group B 0 Before surgery
Weaning from CPB
24 hours after CPB
Comparison vs before operation: * p < 0.05, ** p < 0.01 Comparison vs Group A: ## p < 0.01
Figure 4.
206
Change in cGMP (pmol/ml). CPB = cardiopulmonary bypass.
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EXPRESSION OF IL-Iβ, TNF-α, IL-10 AND cGMP AFTER CPB
inflammatory response, while IL-10 plays a protective role by suppressing inflammatory cytokines.19–21 The mechanism of lung failure after cardiac surgery remains to be defined. Understanding of the factors that control pulmonary vasomotion and the role of NO have increased greatly. NO is the main factor that regulates pulmonary vascular tone.8,9 Its concentration is closely related to the occurrence of PAH. Due to the fact that it is difficult to measure the NO concentration in the body, we measured the cGMP concentration. cGMP level is closely related to NO level.10,11 At baseline, the IL-1β and TNF-α levels in Group A were lower than those in Group B, while the IL-10 level in Group A was higher than that in Group B. This suggests that the balance between proinflammatory and antiinflammatory cytokines at baseline is different among patients with different PAPs. At baseline, patients with PAH may suffer from increased production of IL-1β, TNF-α and decreased production of IL-10. These baseline differences may be the result of abnormally functioning pulmonary arterial endothelium in patients with PAH. Patients with PAH have decreased expression of NOS, which results in decreased release of NO.22 Decreased NO may contribute to the increased levels of proinflammatory cytokines. The increased proinflammatory cytokines also may be related to the pathology of patients undergoing valve replacement surgery and may be responsible for the lower ejection fraction in Group B. We suggest that the balance between proinflammatory response and anti-inflammatory response, which may be important for the clinical outcome, could be different in patients with PAH. The cytokines IL-1β, TNF-α and IL-10 of both groups rose or decreased significantly after CPB (except for TNF-α of Group A). These changes are related to CPB.19–21 The interaction between blood and synthetic surfaces during CPB results in the activation of complement and the coagulation system, neutrophils and leukocytes16,17, 23–25 and induces systemic proinflammatory cytokines2,19–21,26 IL-1β and TNF-α of Group B rose significantly compared with group A after CPB. We believe that IL-1β and TNF-α are more highly expressed after induction in patients with PAH after CPB. Increased TNF-α may contribute to myocardial dysfunction and haemodynamic instability following CPB.27,28 The IL-10 level of Group A remained higher than that of Group B after CPB. We believe that the aggravated inflammatory reaction consumes more IL-10 in patients with PAH. The balance of inflammatory and
Asian Journal of Surgery
anti-inflammatory reactions is upset and is liable to aggravate the inflammatory reaction. The increased levels of IL-1β and TNF-α result in overexpression of adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1, 3–5 which promote leukocyte adhesion, filtration and transmigration, leading to vascular damage.29,30 The level of cGMP was significantly lower in Group B at baseline which implies that the endogenous NO, produced by constitutive NOS, is lower in patients with PAH. cGMP was much lower in Group B after CPB compared to Group A. This may show that NO is produced less after CPB in patients with PAH. Research suggests that NO production significantly increases during and after CPB and is induced by proinflammatory cytokines via the expression of iNOS,12,31 especially in the lungs and airways.32,33 The decrease in NO found in our study may have contributed to the decreased expression of NOS as a consequence of PAH.22 Based on our study, it appears that the baseline level of proinflammatory cytokines IL-1β and TNF-α are higher and the anti-inflammatory cytokine IL-10 is lower in patients with PAH. The baseline level of cGMP was also lower in this group of patients. The production of systemic proinflammatory cytokines IL-1β and TNF-α was highly expressed and the anti-inflammatory cytokine IL-10 was less expressed in patients with PAH compared to patients with normal PAP after CPB. Decreased production of cGMP was also observed in this group of patients. These circumstances may be responsible for postoperative organ dysfunction and morbidity and mortality, especially in patients with PAH. Anti-inflammatory therapy may be a potential method to treat PAH during the perioperative period of cardiac surgery.
ACKNOWLEDGEMENT We thank Dr. Xu X. Hui for his expert laboratory work and Dr. J. M. Luk, of the Department of Surgery of the University of Hong Kong for his critical comments.
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