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Increased Pulmonary Vascular Contraction to Serotonin after Cardiopulmonary Bypass: Role of Cyclooxygenase
Journal of Surgical Research 90, 138 –143 (2000) doi:10.1006/jsre.2000.5869, available online at http://www.idealibrary.com on
Increased Pulmonary Vascular Contraction to Serotonin after Cardiopulmonary Bypass: Role of Cyclooxygenase 1 Kaori Sato, M.D., Jianyi Li, M.S., Caroline Metais, M.D., Cesario Bianchi, M.D., Ph.D., and Frank Sellke, M.D. 2 Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 110 Francis Street, Boston, Massachusetts 02215 Presented at the Annual Meeting of the Association for Academic Surgery, Philadelphia, Pennsylvania, November 18 –20, 1999
Background. Pulmonary vascular resistance is frequently elevated after cardiopulmonary bypass (CPB). We examined if altered pulmonary microvascular reactivity to serotonin (5-HT) is due to altered expression of isoforms of nitric oxide synthase (NOS) or cyclooxygenase (COX). Materials and methods. Pigs (n ⴝ 8) were heparinized and placed on total CPB for 90 min and then perfused off CPB for 90 min. Noninstrumented pigs (n ⴝ 6) served as controls for vascular studies. Relaxation responses (% of precontraction) of microvessels (60 –150 m in diameter) were examined in vitro in a pressurized (20 mm Hg) no-flow state with video microscopic imaging. Expression of eNOS, iNOS, and inducible (COX-2) and constitutive (COX-1) cyclooxygenase was examined with Western blotting and reverse transcription polymerase chain reaction. Results. Pulmonary vascular resistance (PVR) increased from 316 ⴞ 39 mm Hg ⴛ s/cm 5 at baseline to 495 ⴞ 53 at 60 min and 565 ⴞ 62 at 90 min after termination of CPB. 5-HT elicited a relaxation response (46.8 ⴞ 11.8%) in precontracted control microvessels. This response was not affected by the NOS inhibitor N G-nitro-L-arginine. After CPB, pulmonary microvessels contracted significantly to 5-HT (ⴚ29 ⴞ 27%, P < 0.05 vs control). This response was partially inhibited (7 ⴞ 20%, P ⴝ 0.06) in the presence of the COX-2 inhibitor NS398, but was unaffected by the thromboxane synthase inhibitor U63557A (ⴚ20 ⴞ 19%). Expression of iNOS or COX-1 was not changed after CPB. Protein 1
Supported by National Heart, Lung, and Blood Institute Grant HL46716. 2 To whom correspondence and reprint requests should be addressed at Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, 110 Francis Street, LMOB 2A, Boston, MA 02215. Fax: (617) 632-7562. E-mail: [email protected].
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
and mRNA expressions of COX-2 both increased significantly after CPB, while that of eNOS decreased by approximately 50%. Conclusions. PVR increased after CPB. This was associated with a hypercontractile response of isolated pulmonary microvessels to 5-HT that was in part mediated by the release of prostaglandins (but not thromboxane) and associated with increased expression of COX-2 and with decreased expression of eNOS. © 2000 Academic Press Key Words: pulmonary microcirculation; ischemia– reperfusion; serotonin; lung; heart surgery. INTRODUCTION
Increased pulmonary vascular resistance and mismatching of lung ventilation and perfusion are recognized components of respiratory failure after cardiac operations. Total cardiopulmonary bypass (CPB), with diversion of pulmonary blood flow in the absence of ventilation, may in part lead to these pathophysiologic changes. Although the oxygen requirement of pulmonary tissue is not great, a state of reduced pulmonary perfusion followed by a restoration of normal blood flow may contribute to the injury to the lung. In addition, extracorporeal circulation is associated with systemic inflammatory reactions such as complement activation, increased oxidative stress, and neutrophil adhesion and sequestration, all of which may have a detrimental effect on the lungs. Although controversial, the primary site of vascular resistance in the human pulmonary circulation is thought to be at the level of arterioles less than 300 m in diameter [1]. Vascular reactivity and tone are determined by the interaction between neurohumoral substances, vascular smooth muscle, and the endothelium.
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Previous studies have examined the effects of CPB and ischemia–reperfusion on pulmonary vascular dysfunction, but have tended to emphasize the NO pathway. While release of endothelium-derived NO contributes in a major way to the regulation of vasomotor tone, vascular permeability, and other aspects of vascular control, the role of the cyclooxygenase pathway has recently been appreciated as also contributing to pathophysiologic changes during CPB [2], sepsis [3], and other inflammatory states. The purpose of this study was to examine the effects of total CPB, with its associated lack of pulmonary arterial perfusion, on the altered pulmonary vascular response to serotonin, and to define the contribution of isoforms of cyclooxygenase (COX) and NO synthase (NOS) to the altered vascular response. MATERIALS AND METHODS
Animal Preparation Yorkshire pigs (20 –25 kg) of either sex were premedicated with ketamine (10 mg/kg, im) and anesthetized with ␣-chloralose and urethane (60 and 300 mg/kg iv initially, and then 15 and 60 mg/kg every 60 min as needed, respectively). Pigs were intubated and mechanically ventilated (Harvard Apparatus Inc., South Natick, MA). In the baseline control group (n ⫽ 8), sternotomy was performed before the pig was heparinized (500 units/kg). A portion of lung tissue was excised and immediately placed in a cold (4°–10°C) Krebs’ buffer solution of the following composition (in mmol/L): 118.3 NaCl, 4.7 KCl, 2.5 CaCl 2, 1.2 MgSO 4, 1.2 NaH 2PO 4, 25.0 NaHCO 3, 11.1 glucose. Microvessels from this tissue was used for reactivity experiments. Another portion of lung was placed in liquid nitrogen in preparation for molecular studies. In another group of pigs (n ⫽ 8), a sternotomy was performed and pursestring sutures were placed in the distal ascending aorta and the right atrium after heparinization. A flow probe (Transonics System Inc.) was placed around the main pulmonary artery to measure total cardiac output before and after CPB. CPB was instituted using a bubble oxygenator (Bentley Bio-2, Baxter Healthcare Corp., Irvine, CA) and a standard roller pump. An arterial filter (Bentley Bio-1025, Baxter Healthcare Corp.) was inserted into the circuit distal to the roller pump. During CPB, blood flow was maintained from 80 to 100 mL/kg/min to maintain a mean perfusion pressure of 40 –70 mm Hg. Systemic blood temperature was maintained at 37°C during the entire study. Arterial blood gases were obtained (Model 1306, pH/ Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) prior to commencing CPB and at 15-min intervals thereafter. Arterial blood gases were adjusted by ventilatory rate and tidal volume to maintain PAO 2 ⬎ 60 mm Hg, PACO 2 ⬎ 30 and ⬍ 45 mm Hg, and pH between 7.35 and 7.45. After 90 min of total CPB, pigs were separated from bypass and decannulated. Inotropic drugs that could interfere with later vascular experiments were not used during the study. Mean reperfusion pressure was maintained from 40 to 70 mm Hg with fluid resuscitation. After 90 min of post-CPB perfusion, a portion of the lung tissue was excised and immediately placed in cold Krebs’ buffer solution and another portion was placed in liquid nitrogen. All animals received humane care in compliance with the Animal Care and Use Committee of Beth Israel Deaconess Medical Center and the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of
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Science and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1987).
Microvessel Study Protocols Relaxation responses of microvessels were examined after precontraction with the thromboxane A 2 analog U46619 (10 M). Vascular responses to serotonin (5-HT, 1 nM–100 M) and sodium nitroprusside (SNP, 1 nM–100 M) were examined. Selected experiments were performed in the presence of 100 M N G-nitro-L-arginine (LNNA) in the control group and 1 M selective cyclooxygenase 2 (COX-2) inhibitor NS-398 or the thromboxane synthase inhibitor U63557A in post-CPB groups for 20 min. All drugs were applied extraluminally. The vessels were washed three times with Krebs’ buffer solution and allowed to equilibrate in drug-free Krebs’ buffer solution for 15–30 min between drug interventions. Amplification and quantitation of iNOS cDNA fragment by RTPCR. For iNOS mRNA studies, the reverse transcriptase (RT) polymerase chain reaction (PCR) was performed because the intensity of signal for iNOS was not sufficient for quantitative analysis by Northern hybridization. Primers were designed based on the published iNOS sequence [4]. The primer of sense 5⬘-GCCTCGCTCTGGAAAGA-3⬘ corresponding to bases 1425–1441 and the antisense 5⬘-TCCATGCAGACAACCTT-3⬘ corresponding to bases 1908 –1924 were used to amplify a 500-bp fragment of iNOS. An equal amount of total RNA was used for RT-PCR. For quantification, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified from the same amount of RNA to correct for variation of different samples. The PCR products were loaded in 1% agarose gel, then scanned and quantitated using Image-Quant software (Molecular Dynamics, Sunnyvale, CA). Expression of COX-1 and COX-2 (RT-PCR). Primers were designed based on the published COX-1 [5] and COX-2 sequence [6] sequences. For COX-1, the primer of the sense 5⬘-TCTTTGCACAACACTTCACC-3⬘ corresponding to bases 601– 620 and the antisense 5⬘-GTACTCATTGAAGGGCTGCA-3⬘ corresponding to bases 1381–1400 were used to amplify a 799-bp fragment of COX-1. For COX-2, the primer of the sense 5⬘-TAAACTGCGCCTTTTCAAGG-3⬘, corresponding to bases 781– 800, and the antisense 5⬘-GTGATACTTTCTGTACTGCG-3⬘, corresponding to bases 1381–1400, were used to amplify a 619-bp fragment of COX-2. As above, an equal amount of total RNA was used for RT-PCR. For quantification, GAPDH was amplified from the same amount of RNA to correct for variation of different samples. The PCR products were loaded in 1% agarose gel, then scanned and quantitated using Image-Quant software (Molecular Dynamics). Assessment of protein content by Western blotting. Total proteins from pulmonary tissues were obtained by homogenizing in a lysis buffer containing 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) and centrifuging at 12,000g for 10 min at 4°C. Protein concentration of the supernatant was measured by spectrophotometry at 595 nm (DU640, Beckman, Fullerton, CA) of an aliquot developed for 10 min in Protein Assay Dye Reagent (Bio-Rad, Hercules, CA). Total protein (40 g/lane) was fractionated on 10% SDS–polyacrylamide gel transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). Equal protein loading and transfer efficiency was visualized by Ponceau red staining. The membrane was incubated with 5% nonfat dry milk powder, 0.05% Tween-20 in phosphate-buffered saline (PBS) for 12 h at 4°C to block for nonspecific absorption, then was immunoblotted with the monoclonal mouse anti-endothelial NOS antibody (Transduction Laboratories, Lexington, KY) 1:2500 (v/v) dilution or the monoclonal mouse antiinducible NOS antibody (Transduction Laboratories) 1:500 dilution (v/v) for 2 h for eNOS and iNOS Western, or with the polyclonal goat anti-COX1 (Santa Cruz) 1/1000 dilution (v/v) or the polyclonal goat-anti COX2 1/500 dilution antibody (Santa Cruz). After washing with PBS, the membrane was
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FIG. 1. Plot of in vitro response of precontracted porcine pulmonary arterioles to 5-HT. Vessels were harvested from the lungs before (control, n ⫽ 8) and after (CPB-Rep, n ⫽ 8) CPB-reperfusion. Selected experiments were performed in the presence of the NOS inhibitor N G-nitro-L-arginine in the control group or the cyclooxygenase-2 inhibitor NS398 or the thromboxane synthase inhibitor U63557A in the CPB-reperfusion group. Responses are percentage change in diameter of degree of precontraction. Data are expressed as means ⫾ SEM. Two-way ANOVA for repeated measures. *P ⬍ 0.05 versus control.
incubated for 1 h in 5% milk powder PBS containing 1:3000 diluted goat anti-mouse IgG conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) or anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz). Peroxidase activity was visualized using an enhanced chemiluminescence substrate system (Amersham, Arlington Heights, IL). Densitometry of digitized images of immunoprobed membraned (ScanJet 4c, Hewlett–Packard) was performed using Image Quant software (Molecular Dynamics).
Vessel Characteristics Pulmonary microvessels ranged from 60 to 150 m in internal diameter, averaging 128 ⫾ 7 m. Percentage precontraction after spontaneous constriction and/or after application of the thromboxane A 2 analog was 28 ⫾ 3% in the control group and 29 ⫾ 4% in the CPB-reperfusion group.
Drugs U46619, sodium nitroprusside, and serotonin were obtained from Sigma (St. Louis, MO). N G-Nitro-L-arginine, NS-398, and U63557A were obtained from RBI (Natick, MA). All other drugs were dissolved in ultrapure distilled water and prepared on the day of the study.
Data Analysis The relaxation responses were expressed as the percentage relaxation (mean ⫾ SEM) of the U46619-induced precontraction. Comparisons of dose–response curves between experimental groups were performed by one- or two-way analysis of variance for repeated measurements, followed by Fisher’s LSD test and the Bonferroni correction when indicated. Student’s t test was used to compare changes in hemodynamic variables and for gene and protein expression. Statistical significance was taken at P ⬍ 0.05.
RESULTS
Prior to cardiopulmonary bypass, mean pulmonary artery pressure (MPA, mm Hg) and pulmonary vascular resistance (PVR, dyn ⫻ s/cm 5) were 14.2 ⫾ 2.0 and 316 ⫾ 32, respectively. These increased to 21.6 ⫾ 1.8 and 504 ⫾ 96 immediately after CPB and to 15 ⫾ 3 and 565 ⫾ 62 after 90 min of post-CPB perfusion, respectively.
In Vitro Response to 5-HT 5-HT, a platelet-derived substance that produces both receptor-mediated endothelium-dependent relaxation and direct vascular smooth muscle contraction, had a minimal net effect on control microvessels. Surprisingly, this response was not affected in the presence of L-NNA. In contrast, 5-HT elicited a marked contraction after CPB-reperfusion. This contraction was reduced (P ⫽ 0.06) but not totally inhibited in the presence of the selective cyclooxygenase 2 inhibitor NS-398. The increased contractile response was not significantly affected by the thromboxane synthase inhibitor U63557A, suggesting that a substance other than thromboxane mediates this effect (Fig. 1). In Vitro Response to SNP Sodium nitroprusside, a direct vascular smooth muscle relaxation agent, elicited similar relaxation before and after CPB (Fig. 2). This suggests that the ability of the vascular smooth muscle to relax to a cyclic GMPmediated agonist was unchanged by CPB.
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FIG. 2. Plot of in vitro response of precontracted porcine pulmonary arterioles to SNP. Vessels were harvested from the lungs before (control, n ⫽ 8) and after (CPB-Rep, n ⫽ 8) CPBreperfusion. Responses are percentage change in diameter of degree of precontraction.
Gene Expression of iNOS, COX-1, and COX-2 To examine whether the endothelium dysfunction observed after CPB is due to an altered expression of the inducible isoform of NOS or to enzymes responsible for synthesis of prostaglandins, expression of iNOS, COX-1, and COX-2 was analyzed by RT-PCR. Gene expression of iNOS as well as expression of COX-1 was not altered after CPB. In contrast, expression of COX-2 was significantly increased by 30 ⫾ 7% (P ⬍ 0.05) after CPB-reperfusion (Fig. 3).
FIG. 4. (A) Representative Western blot products of constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in porcine lung tissue before (control, n ⫽ 4) and after (CPB-Rep, n ⫽ 4) CPB-reperfusion. (B) Representative Western blot products of constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in porcine lung tissue before (control, n ⫽ 4) and after (CPB-Rep, n ⫽ 4) CPB-reperfusion. One microgram of lung tissue was subjected to reverse transcription reaction and reaction mixtures were amplified by polymerase chain reaction and then analyzed by agarose gel expression.
Protein Content of eNOS, iNOS, COX-1, and COX-2 Similar to what was observed with RT-PCR, Western analysis of protein content demonstrated increased 2.0 ⫾ 0.3-fold (P ⬍ 0.01) protein levels of COX-2 in the pulmonary tissue after CPB. Protein content of eNOS was decreased 40 ⫾ 9% (P ⬍ 0.05) after CPB. However, protein contents of iNOS and COX-1 were similar before and after CPB (Fig. 4). FIG. 3. Constitutive cyclooxygenase (COX-1), inducible cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in porcine lung tissue before (control, n ⫽ 4) and after (CP-Rep, n ⫽ 4) CPB-reperfusion. One microgram of lung tissue was subjected to reverse transcription reaction and reaction mixtures were amplified by polymerase chain reaction and then analyzed by agarose gel expression. Positions and sizes of the DNA markers [in base pairs (bp)] are shown at the right.
DISCUSSION
The major finding of the present study is that total cardiopulmonary bypass followed with restoration of normal perfusion markedly alters pulmonary microvascular responses to the platelet-derived substance 5-HT. This enhanced microvascular response to 5-HT
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is due in part to an increased production and release of products of COX, other than thromboxane A 2. Since this contractile response elicited by 5-HT was partly reduced in the presence of a selective inhibitor of COX-2 and the 5-HT induced response was not significantly affected by L-NNA, it is likely that an altered expression or activity of COX may account, at least in large part, for this enhanced contraction to 5-HT. Previously we have demonstrated that the hypercontractile response of sheep pulmonary microvessels is inhibited by a nonselective COX inhibitor, indomethacin [10]. It was somewhat surprising that the selective COX-2 inhibitor did not normalize the response to 5-HT. This failure to completely normalize the response may be due to incomplete inhibition of COX-2 by NS-398, or it may be the case that products of the constitutive COX-1 are involved in the enhanced contractile response. The response to an endotheliumindependent agonist sodium nitroprusside was not changed after CPB, suggesting that the ability of the vascular smooth muscle to relax was not affected. The observation that COX-2 seems to be regulated by agonist stimulation is somewhat surprising since the NO producing counterpart iNOS is not regulated by agonists. Prostaglandins are formed by the action of isoforms of COX in a two-step conversion of arachidonic acid. 5-HT causes the release of arachidonic acid metabolites from the plasma membrane, which probably accounts for the increased activity of COX-2 to agonist stimulation. COX converts arachidonic acid to a cyclic endoperoxide. This cyclic endoperoxide is cleaved off the peroxide to yield to an endoperoxide (PGH 2). These unstable intermediate products of arachidonic acid metabolism are rapidly converted to the prostaglandins by specific isomerase enzymes [7]. Both COX-1 and COX-2 use the same endogenous substrate, arachidonic acid, and form the same product by the same catalytic mechanism; their major difference lies in their pathological functions. In inflammatory processes, COX-2 is expressed in many cells including fibroblasts and macrophages, and accounts for the release of large quantities of proinflammatory prostaglandins at the site of inflammation [8]. In addition, COX-2 is contitutively expressed in lung and gut tissue. Which prostaglandin products predominate depends largely on the relative activity of the two isoforms of COX but also on the secondary pathways that yield the different prostanoids. Increased oxidative stress and other conditions in the tissue can influence which prostaglandins are synthesized. It is likely that both isoforms of COX contribute to the enhanced contractile response to 5-HT after CPB. A reduced relaxation elicited by substance P, acetylcholine, and other endothelium-dependent agonists after CPB has been reported and is probably due to a
reduction in the release of nitric oxide [9]. Thus, both NOS and COX vasoregulatory pathways are altered in the porcine pulmonary circulation. However, in the porcine pulmonary circulation, 5-HT did not appear to elicit the release of NO. Cardiac operations in which cardiopulmonary bypass is used elicit a systemic inflammatory response that can contribute to organ injury and postoperative morbidity. Causative factors include surgical trauma, contact of blood with the extracorporeal circuit, and lung reperfusion injury on discontinuation of bypass. In addition, CPB leads to the activation of the complement, coagulation, fibrinolytic, and kallikrein cascades, activation of neutrophils with degranulation and protease enzyme release, oxygen radical production, and the synthesis of various cytokines from mononuclear cells including tumor necrosis factor ␣, interleukin (IL)-6, Il-1, and other cytokines are important components of inflammation and the immune response [10, 11]. The increased expression of COX-2 may be due to both systemic and local factors. Increased production of Il-1 has been transiently found after CPB, reaching a maximum at 24 h [12]. Il-1 induces the expression of the COX-2 mRNA as early as 15 min after exposure [13]. It is probable that other inflammatory cytokines play a similar role in causing these changes in vasomotor regulation. CLINICAL IMPLICATIONS
Since pulmonary hypertension and right-sided heart failure are not uncommon following cardiac operations in which cardiopulmonary bypass is used, altered vasomotor tone of pulmonary resistance vessels may have considerable impact on postoperative recovery. The present study demonstrates altered endotheliumdependent pulmonary microvascular responses to 5-HT after total CPB. The role of serotonergic and other endothelium-dependent mechanisms in regulating ventilation–perfusion is not known. However, it is possible that this altered pattern of gene expression and the associated pattern of vascular regulation may disrupt normal pulmonary flow and lead to ventilation–perfusion mismatch, shunting, lung edema and other aspects of pulmonary dysfunction after CPB. REFERENCES 1.
Kato, M., and Staub, N. C. Response of small pulmonary arteries to unilobular hypoxia and hypercapnia. Circ. Res. 19: 426, 1966. 2. Metais, C., Li, J. Y., Li, J., Simons, M., and Sellke, F. W. Serotonin-induced coronary contraction increases after blood cardioplegia–reperfusion: Role of COX-2 expression. Circulation 100: II-328, 1999. 3. Sellke, F. W. The role of cyclooxygenase in regulation of vasomotor tone during endotoxemia. Shock 11: 443, 1999.
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Geller, D. A., Lowenstein, C. J., Shapiro, R. A., Nussler, A. K., Di Silvio, M., Wang, J. C., Nakayama, D. K., Simons, R. L., Synder, S. H., and Billiar, T. R. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. USA. 90: 3491, 1993. Funk, C. D., Funk, L. B., Kennedy, M. E., Pong, A. S., and Fitzgerald, G. A. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression and gene chromosomal assignment. FASEB J. 5: 2304, 1991. Hla, T., and Neilson, K. Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA 89: 7384, 1992. Needleman, P., Turk, J., Jakschik, B. A., Morrison, A. R., and Lefkowith, J. B. Arachidonic acid metabolism. Annu. Rev. Biochem. 55: 69, 1986. Wu, K. K. Inducible cyclooxygenase and nitric oxide synthase. Adv. Pharmacol. 33: 179, 1995.
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Shafique, T., Johnson, R. G., Dai, H. B., Weintraub, R. M., and Sellke, F. W. Altered pulmonary microvascular reactivity following total cardiopulmonary bypass. J. Thorac. Cardiovasc. Surgery 105: 479, 1993. Dinarello, C. A. Interleukin-1 and interleukin-1 antagonism. Blood 77: 1627, 1991. Dinarello, C., and Wolff, S. M. The role of interleukin-1 in disease. N. Engl. J. Med. 328: 106, 1993. Haeffner-Cavaillon, N., Rousselier, N., Ponzio, O., Carreno, M. P., Laude, M., Carpentier, A., and Kazatchkine, M. D. Induction of interleukin-1 production in patients undergoing cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg. 98: 1100, 1989. Ristimaki, A., Garfinkel, S., Wessendorf, J., Maciag, T. and Hla, T. Induction of cyclooxygenase-2 by interleukin-1 alpha. J. Biol. Chem. 269: 11769, 1994.
Increased Pulmonary Vascular Contraction to Serotonin after Cardiopulmonary Bypass: Role of Cyclooxygenase 1 Kaori Sato, M.D., Jianyi Li, M.S., Caroline Metais, M.D., Cesario Bianchi, M.D., Ph.D., and Frank Sellke, M.D. 2 Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 110 Francis Street, Boston, Massachusetts 02215 Presented at the Annual Meeting of the Association for Academic Surgery, Philadelphia, Pennsylvania, November 18 –20, 1999
Background. Pulmonary vascular resistance is frequently elevated after cardiopulmonary bypass (CPB). We examined if altered pulmonary microvascular reactivity to serotonin (5-HT) is due to altered expression of isoforms of nitric oxide synthase (NOS) or cyclooxygenase (COX). Materials and methods. Pigs (n ⴝ 8) were heparinized and placed on total CPB for 90 min and then perfused off CPB for 90 min. Noninstrumented pigs (n ⴝ 6) served as controls for vascular studies. Relaxation responses (% of precontraction) of microvessels (60 –150 m in diameter) were examined in vitro in a pressurized (20 mm Hg) no-flow state with video microscopic imaging. Expression of eNOS, iNOS, and inducible (COX-2) and constitutive (COX-1) cyclooxygenase was examined with Western blotting and reverse transcription polymerase chain reaction. Results. Pulmonary vascular resistance (PVR) increased from 316 ⴞ 39 mm Hg ⴛ s/cm 5 at baseline to 495 ⴞ 53 at 60 min and 565 ⴞ 62 at 90 min after termination of CPB. 5-HT elicited a relaxation response (46.8 ⴞ 11.8%) in precontracted control microvessels. This response was not affected by the NOS inhibitor N G-nitro-L-arginine. After CPB, pulmonary microvessels contracted significantly to 5-HT (ⴚ29 ⴞ 27%, P < 0.05 vs control). This response was partially inhibited (7 ⴞ 20%, P ⴝ 0.06) in the presence of the COX-2 inhibitor NS398, but was unaffected by the thromboxane synthase inhibitor U63557A (ⴚ20 ⴞ 19%). Expression of iNOS or COX-1 was not changed after CPB. Protein 1
Supported by National Heart, Lung, and Blood Institute Grant HL46716. 2 To whom correspondence and reprint requests should be addressed at Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, 110 Francis Street, LMOB 2A, Boston, MA 02215. Fax: (617) 632-7562. E-mail: [email protected].
0022-4804/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
and mRNA expressions of COX-2 both increased significantly after CPB, while that of eNOS decreased by approximately 50%. Conclusions. PVR increased after CPB. This was associated with a hypercontractile response of isolated pulmonary microvessels to 5-HT that was in part mediated by the release of prostaglandins (but not thromboxane) and associated with increased expression of COX-2 and with decreased expression of eNOS. © 2000 Academic Press Key Words: pulmonary microcirculation; ischemia– reperfusion; serotonin; lung; heart surgery. INTRODUCTION
Increased pulmonary vascular resistance and mismatching of lung ventilation and perfusion are recognized components of respiratory failure after cardiac operations. Total cardiopulmonary bypass (CPB), with diversion of pulmonary blood flow in the absence of ventilation, may in part lead to these pathophysiologic changes. Although the oxygen requirement of pulmonary tissue is not great, a state of reduced pulmonary perfusion followed by a restoration of normal blood flow may contribute to the injury to the lung. In addition, extracorporeal circulation is associated with systemic inflammatory reactions such as complement activation, increased oxidative stress, and neutrophil adhesion and sequestration, all of which may have a detrimental effect on the lungs. Although controversial, the primary site of vascular resistance in the human pulmonary circulation is thought to be at the level of arterioles less than 300 m in diameter [1]. Vascular reactivity and tone are determined by the interaction between neurohumoral substances, vascular smooth muscle, and the endothelium.
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Previous studies have examined the effects of CPB and ischemia–reperfusion on pulmonary vascular dysfunction, but have tended to emphasize the NO pathway. While release of endothelium-derived NO contributes in a major way to the regulation of vasomotor tone, vascular permeability, and other aspects of vascular control, the role of the cyclooxygenase pathway has recently been appreciated as also contributing to pathophysiologic changes during CPB [2], sepsis [3], and other inflammatory states. The purpose of this study was to examine the effects of total CPB, with its associated lack of pulmonary arterial perfusion, on the altered pulmonary vascular response to serotonin, and to define the contribution of isoforms of cyclooxygenase (COX) and NO synthase (NOS) to the altered vascular response. MATERIALS AND METHODS
Animal Preparation Yorkshire pigs (20 –25 kg) of either sex were premedicated with ketamine (10 mg/kg, im) and anesthetized with ␣-chloralose and urethane (60 and 300 mg/kg iv initially, and then 15 and 60 mg/kg every 60 min as needed, respectively). Pigs were intubated and mechanically ventilated (Harvard Apparatus Inc., South Natick, MA). In the baseline control group (n ⫽ 8), sternotomy was performed before the pig was heparinized (500 units/kg). A portion of lung tissue was excised and immediately placed in a cold (4°–10°C) Krebs’ buffer solution of the following composition (in mmol/L): 118.3 NaCl, 4.7 KCl, 2.5 CaCl 2, 1.2 MgSO 4, 1.2 NaH 2PO 4, 25.0 NaHCO 3, 11.1 glucose. Microvessels from this tissue was used for reactivity experiments. Another portion of lung was placed in liquid nitrogen in preparation for molecular studies. In another group of pigs (n ⫽ 8), a sternotomy was performed and pursestring sutures were placed in the distal ascending aorta and the right atrium after heparinization. A flow probe (Transonics System Inc.) was placed around the main pulmonary artery to measure total cardiac output before and after CPB. CPB was instituted using a bubble oxygenator (Bentley Bio-2, Baxter Healthcare Corp., Irvine, CA) and a standard roller pump. An arterial filter (Bentley Bio-1025, Baxter Healthcare Corp.) was inserted into the circuit distal to the roller pump. During CPB, blood flow was maintained from 80 to 100 mL/kg/min to maintain a mean perfusion pressure of 40 –70 mm Hg. Systemic blood temperature was maintained at 37°C during the entire study. Arterial blood gases were obtained (Model 1306, pH/ Blood Gas Analyzer, Instrumentation Laboratory, Lexington, MA) prior to commencing CPB and at 15-min intervals thereafter. Arterial blood gases were adjusted by ventilatory rate and tidal volume to maintain PAO 2 ⬎ 60 mm Hg, PACO 2 ⬎ 30 and ⬍ 45 mm Hg, and pH between 7.35 and 7.45. After 90 min of total CPB, pigs were separated from bypass and decannulated. Inotropic drugs that could interfere with later vascular experiments were not used during the study. Mean reperfusion pressure was maintained from 40 to 70 mm Hg with fluid resuscitation. After 90 min of post-CPB perfusion, a portion of the lung tissue was excised and immediately placed in cold Krebs’ buffer solution and another portion was placed in liquid nitrogen. All animals received humane care in compliance with the Animal Care and Use Committee of Beth Israel Deaconess Medical Center and the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of
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Science and published by the National Institutes of Health (NIH Publication No. 80-23, revised 1987).
Microvessel Study Protocols Relaxation responses of microvessels were examined after precontraction with the thromboxane A 2 analog U46619 (10 M). Vascular responses to serotonin (5-HT, 1 nM–100 M) and sodium nitroprusside (SNP, 1 nM–100 M) were examined. Selected experiments were performed in the presence of 100 M N G-nitro-L-arginine (LNNA) in the control group and 1 M selective cyclooxygenase 2 (COX-2) inhibitor NS-398 or the thromboxane synthase inhibitor U63557A in post-CPB groups for 20 min. All drugs were applied extraluminally. The vessels were washed three times with Krebs’ buffer solution and allowed to equilibrate in drug-free Krebs’ buffer solution for 15–30 min between drug interventions. Amplification and quantitation of iNOS cDNA fragment by RTPCR. For iNOS mRNA studies, the reverse transcriptase (RT) polymerase chain reaction (PCR) was performed because the intensity of signal for iNOS was not sufficient for quantitative analysis by Northern hybridization. Primers were designed based on the published iNOS sequence [4]. The primer of sense 5⬘-GCCTCGCTCTGGAAAGA-3⬘ corresponding to bases 1425–1441 and the antisense 5⬘-TCCATGCAGACAACCTT-3⬘ corresponding to bases 1908 –1924 were used to amplify a 500-bp fragment of iNOS. An equal amount of total RNA was used for RT-PCR. For quantification, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified from the same amount of RNA to correct for variation of different samples. The PCR products were loaded in 1% agarose gel, then scanned and quantitated using Image-Quant software (Molecular Dynamics, Sunnyvale, CA). Expression of COX-1 and COX-2 (RT-PCR). Primers were designed based on the published COX-1 [5] and COX-2 sequence [6] sequences. For COX-1, the primer of the sense 5⬘-TCTTTGCACAACACTTCACC-3⬘ corresponding to bases 601– 620 and the antisense 5⬘-GTACTCATTGAAGGGCTGCA-3⬘ corresponding to bases 1381–1400 were used to amplify a 799-bp fragment of COX-1. For COX-2, the primer of the sense 5⬘-TAAACTGCGCCTTTTCAAGG-3⬘, corresponding to bases 781– 800, and the antisense 5⬘-GTGATACTTTCTGTACTGCG-3⬘, corresponding to bases 1381–1400, were used to amplify a 619-bp fragment of COX-2. As above, an equal amount of total RNA was used for RT-PCR. For quantification, GAPDH was amplified from the same amount of RNA to correct for variation of different samples. The PCR products were loaded in 1% agarose gel, then scanned and quantitated using Image-Quant software (Molecular Dynamics). Assessment of protein content by Western blotting. Total proteins from pulmonary tissues were obtained by homogenizing in a lysis buffer containing 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) and centrifuging at 12,000g for 10 min at 4°C. Protein concentration of the supernatant was measured by spectrophotometry at 595 nm (DU640, Beckman, Fullerton, CA) of an aliquot developed for 10 min in Protein Assay Dye Reagent (Bio-Rad, Hercules, CA). Total protein (40 g/lane) was fractionated on 10% SDS–polyacrylamide gel transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Bedford, MA). Equal protein loading and transfer efficiency was visualized by Ponceau red staining. The membrane was incubated with 5% nonfat dry milk powder, 0.05% Tween-20 in phosphate-buffered saline (PBS) for 12 h at 4°C to block for nonspecific absorption, then was immunoblotted with the monoclonal mouse anti-endothelial NOS antibody (Transduction Laboratories, Lexington, KY) 1:2500 (v/v) dilution or the monoclonal mouse antiinducible NOS antibody (Transduction Laboratories) 1:500 dilution (v/v) for 2 h for eNOS and iNOS Western, or with the polyclonal goat anti-COX1 (Santa Cruz) 1/1000 dilution (v/v) or the polyclonal goat-anti COX2 1/500 dilution antibody (Santa Cruz). After washing with PBS, the membrane was
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FIG. 1. Plot of in vitro response of precontracted porcine pulmonary arterioles to 5-HT. Vessels were harvested from the lungs before (control, n ⫽ 8) and after (CPB-Rep, n ⫽ 8) CPB-reperfusion. Selected experiments were performed in the presence of the NOS inhibitor N G-nitro-L-arginine in the control group or the cyclooxygenase-2 inhibitor NS398 or the thromboxane synthase inhibitor U63557A in the CPB-reperfusion group. Responses are percentage change in diameter of degree of precontraction. Data are expressed as means ⫾ SEM. Two-way ANOVA for repeated measures. *P ⬍ 0.05 versus control.
incubated for 1 h in 5% milk powder PBS containing 1:3000 diluted goat anti-mouse IgG conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA) or anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz). Peroxidase activity was visualized using an enhanced chemiluminescence substrate system (Amersham, Arlington Heights, IL). Densitometry of digitized images of immunoprobed membraned (ScanJet 4c, Hewlett–Packard) was performed using Image Quant software (Molecular Dynamics).
Vessel Characteristics Pulmonary microvessels ranged from 60 to 150 m in internal diameter, averaging 128 ⫾ 7 m. Percentage precontraction after spontaneous constriction and/or after application of the thromboxane A 2 analog was 28 ⫾ 3% in the control group and 29 ⫾ 4% in the CPB-reperfusion group.
Drugs U46619, sodium nitroprusside, and serotonin were obtained from Sigma (St. Louis, MO). N G-Nitro-L-arginine, NS-398, and U63557A were obtained from RBI (Natick, MA). All other drugs were dissolved in ultrapure distilled water and prepared on the day of the study.
Data Analysis The relaxation responses were expressed as the percentage relaxation (mean ⫾ SEM) of the U46619-induced precontraction. Comparisons of dose–response curves between experimental groups were performed by one- or two-way analysis of variance for repeated measurements, followed by Fisher’s LSD test and the Bonferroni correction when indicated. Student’s t test was used to compare changes in hemodynamic variables and for gene and protein expression. Statistical significance was taken at P ⬍ 0.05.
RESULTS
Prior to cardiopulmonary bypass, mean pulmonary artery pressure (MPA, mm Hg) and pulmonary vascular resistance (PVR, dyn ⫻ s/cm 5) were 14.2 ⫾ 2.0 and 316 ⫾ 32, respectively. These increased to 21.6 ⫾ 1.8 and 504 ⫾ 96 immediately after CPB and to 15 ⫾ 3 and 565 ⫾ 62 after 90 min of post-CPB perfusion, respectively.
In Vitro Response to 5-HT 5-HT, a platelet-derived substance that produces both receptor-mediated endothelium-dependent relaxation and direct vascular smooth muscle contraction, had a minimal net effect on control microvessels. Surprisingly, this response was not affected in the presence of L-NNA. In contrast, 5-HT elicited a marked contraction after CPB-reperfusion. This contraction was reduced (P ⫽ 0.06) but not totally inhibited in the presence of the selective cyclooxygenase 2 inhibitor NS-398. The increased contractile response was not significantly affected by the thromboxane synthase inhibitor U63557A, suggesting that a substance other than thromboxane mediates this effect (Fig. 1). In Vitro Response to SNP Sodium nitroprusside, a direct vascular smooth muscle relaxation agent, elicited similar relaxation before and after CPB (Fig. 2). This suggests that the ability of the vascular smooth muscle to relax to a cyclic GMPmediated agonist was unchanged by CPB.
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FIG. 2. Plot of in vitro response of precontracted porcine pulmonary arterioles to SNP. Vessels were harvested from the lungs before (control, n ⫽ 8) and after (CPB-Rep, n ⫽ 8) CPBreperfusion. Responses are percentage change in diameter of degree of precontraction.
Gene Expression of iNOS, COX-1, and COX-2 To examine whether the endothelium dysfunction observed after CPB is due to an altered expression of the inducible isoform of NOS or to enzymes responsible for synthesis of prostaglandins, expression of iNOS, COX-1, and COX-2 was analyzed by RT-PCR. Gene expression of iNOS as well as expression of COX-1 was not altered after CPB. In contrast, expression of COX-2 was significantly increased by 30 ⫾ 7% (P ⬍ 0.05) after CPB-reperfusion (Fig. 3).
FIG. 4. (A) Representative Western blot products of constitutive nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) in porcine lung tissue before (control, n ⫽ 4) and after (CPB-Rep, n ⫽ 4) CPB-reperfusion. (B) Representative Western blot products of constitutive cyclooxygenase (COX-1) and inducible cyclooxygenase (COX-2) in porcine lung tissue before (control, n ⫽ 4) and after (CPB-Rep, n ⫽ 4) CPB-reperfusion. One microgram of lung tissue was subjected to reverse transcription reaction and reaction mixtures were amplified by polymerase chain reaction and then analyzed by agarose gel expression.
Protein Content of eNOS, iNOS, COX-1, and COX-2 Similar to what was observed with RT-PCR, Western analysis of protein content demonstrated increased 2.0 ⫾ 0.3-fold (P ⬍ 0.01) protein levels of COX-2 in the pulmonary tissue after CPB. Protein content of eNOS was decreased 40 ⫾ 9% (P ⬍ 0.05) after CPB. However, protein contents of iNOS and COX-1 were similar before and after CPB (Fig. 4). FIG. 3. Constitutive cyclooxygenase (COX-1), inducible cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in porcine lung tissue before (control, n ⫽ 4) and after (CP-Rep, n ⫽ 4) CPB-reperfusion. One microgram of lung tissue was subjected to reverse transcription reaction and reaction mixtures were amplified by polymerase chain reaction and then analyzed by agarose gel expression. Positions and sizes of the DNA markers [in base pairs (bp)] are shown at the right.
DISCUSSION
The major finding of the present study is that total cardiopulmonary bypass followed with restoration of normal perfusion markedly alters pulmonary microvascular responses to the platelet-derived substance 5-HT. This enhanced microvascular response to 5-HT
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is due in part to an increased production and release of products of COX, other than thromboxane A 2. Since this contractile response elicited by 5-HT was partly reduced in the presence of a selective inhibitor of COX-2 and the 5-HT induced response was not significantly affected by L-NNA, it is likely that an altered expression or activity of COX may account, at least in large part, for this enhanced contraction to 5-HT. Previously we have demonstrated that the hypercontractile response of sheep pulmonary microvessels is inhibited by a nonselective COX inhibitor, indomethacin [10]. It was somewhat surprising that the selective COX-2 inhibitor did not normalize the response to 5-HT. This failure to completely normalize the response may be due to incomplete inhibition of COX-2 by NS-398, or it may be the case that products of the constitutive COX-1 are involved in the enhanced contractile response. The response to an endotheliumindependent agonist sodium nitroprusside was not changed after CPB, suggesting that the ability of the vascular smooth muscle to relax was not affected. The observation that COX-2 seems to be regulated by agonist stimulation is somewhat surprising since the NO producing counterpart iNOS is not regulated by agonists. Prostaglandins are formed by the action of isoforms of COX in a two-step conversion of arachidonic acid. 5-HT causes the release of arachidonic acid metabolites from the plasma membrane, which probably accounts for the increased activity of COX-2 to agonist stimulation. COX converts arachidonic acid to a cyclic endoperoxide. This cyclic endoperoxide is cleaved off the peroxide to yield to an endoperoxide (PGH 2). These unstable intermediate products of arachidonic acid metabolism are rapidly converted to the prostaglandins by specific isomerase enzymes [7]. Both COX-1 and COX-2 use the same endogenous substrate, arachidonic acid, and form the same product by the same catalytic mechanism; their major difference lies in their pathological functions. In inflammatory processes, COX-2 is expressed in many cells including fibroblasts and macrophages, and accounts for the release of large quantities of proinflammatory prostaglandins at the site of inflammation [8]. In addition, COX-2 is contitutively expressed in lung and gut tissue. Which prostaglandin products predominate depends largely on the relative activity of the two isoforms of COX but also on the secondary pathways that yield the different prostanoids. Increased oxidative stress and other conditions in the tissue can influence which prostaglandins are synthesized. It is likely that both isoforms of COX contribute to the enhanced contractile response to 5-HT after CPB. A reduced relaxation elicited by substance P, acetylcholine, and other endothelium-dependent agonists after CPB has been reported and is probably due to a
reduction in the release of nitric oxide [9]. Thus, both NOS and COX vasoregulatory pathways are altered in the porcine pulmonary circulation. However, in the porcine pulmonary circulation, 5-HT did not appear to elicit the release of NO. Cardiac operations in which cardiopulmonary bypass is used elicit a systemic inflammatory response that can contribute to organ injury and postoperative morbidity. Causative factors include surgical trauma, contact of blood with the extracorporeal circuit, and lung reperfusion injury on discontinuation of bypass. In addition, CPB leads to the activation of the complement, coagulation, fibrinolytic, and kallikrein cascades, activation of neutrophils with degranulation and protease enzyme release, oxygen radical production, and the synthesis of various cytokines from mononuclear cells including tumor necrosis factor ␣, interleukin (IL)-6, Il-1, and other cytokines are important components of inflammation and the immune response [10, 11]. The increased expression of COX-2 may be due to both systemic and local factors. Increased production of Il-1 has been transiently found after CPB, reaching a maximum at 24 h [12]. Il-1 induces the expression of the COX-2 mRNA as early as 15 min after exposure [13]. It is probable that other inflammatory cytokines play a similar role in causing these changes in vasomotor regulation. CLINICAL IMPLICATIONS
Since pulmonary hypertension and right-sided heart failure are not uncommon following cardiac operations in which cardiopulmonary bypass is used, altered vasomotor tone of pulmonary resistance vessels may have considerable impact on postoperative recovery. The present study demonstrates altered endotheliumdependent pulmonary microvascular responses to 5-HT after total CPB. The role of serotonergic and other endothelium-dependent mechanisms in regulating ventilation–perfusion is not known. However, it is possible that this altered pattern of gene expression and the associated pattern of vascular regulation may disrupt normal pulmonary flow and lead to ventilation–perfusion mismatch, shunting, lung edema and other aspects of pulmonary dysfunction after CPB. REFERENCES 1.
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Shafique, T., Johnson, R. G., Dai, H. B., Weintraub, R. M., and Sellke, F. W. Altered pulmonary microvascular reactivity following total cardiopulmonary bypass. J. Thorac. Cardiovasc. Surgery 105: 479, 1993. Dinarello, C. A. Interleukin-1 and interleukin-1 antagonism. Blood 77: 1627, 1991. Dinarello, C., and Wolff, S. M. The role of interleukin-1 in disease. N. Engl. J. Med. 328: 106, 1993. Haeffner-Cavaillon, N., Rousselier, N., Ponzio, O., Carreno, M. P., Laude, M., Carpentier, A., and Kazatchkine, M. D. Induction of interleukin-1 production in patients undergoing cardiopulmonary bypass. J. Thorac. Cardiovasc. Surg. 98: 1100, 1989. Ristimaki, A., Garfinkel, S., Wessendorf, J., Maciag, T. and Hla, T. Induction of cyclooxygenase-2 by interleukin-1 alpha. J. Biol. Chem. 269: 11769, 1994.