Mesenteric dysfunction after cardiopulmonary bypass: role of complement C5a

Mesenteric Dysfunction After Cardiopulmonary Bypass: Role of Complement C5a Motohisa Tofukuji, MD, PhD, Gregory L. Stahl, PhD, Caroline Metais, MD, Mikio Tomita, PhD, Azin Agah, PhD, Cesario Bianchi, MD, PhD, Mitchell P. Fink, MD, and Frank W. Sellke, MD Department of Surgery, Beth Israel-Deaconess Medical Center, and Center For Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts

Background. We investigated the effects of cardiopulmonary bypass (CPB) on ileal homeostasis, and the influence of functional inhibition of complement C5a on CPB-induced mesenteric injury. Methods. Pigs were perfused on CPB for 1 hour and then perfused off CPB for an additional 2 hours. Antiporcine C5a monoclonal antibody (C5a MAb) was administered 20 minutes before onset of CPB to 6 pigs; 6 controls received saline vehicle. Total complement activity, ileal myeloperoxidase, and indices of ileal integrity were examined. Results. Treatment with C5a MAb ameliorated CPBinduced abnormalities in endothelium-dependent relaxation to ADP and substance P, and the hypercontractile response to phenylephrine of ileal microvessels (88 to 168 ␮m). Ileal myeloperoxidase activity [units/g protein] was 41 ⴞ 11 in the C5a MAb group, compared to 83 ⴞ 13

in the saline group (19 ⴞ 10 base line). Total hemolytic complement activity was similar in the C5a MAb and saline groups (0.6 ⴞ 0.2 and 0.7 ⴞ 0.2 CH50 units). During CPB, ileal mucosal blood flow and mucosal pH, edema formation, and epithelial permeability deteriorated similarly in saline and C5a MAb groups. Inducible nitric oxide synthase (iNOS) mRNA expression was similar before and after CPB. Conclusions. CPB is associated with significant physiologic alterations in mucosal perfusion, epithelial permeability, edema formation, and blood flow regulation. Inhibition of C5a limits neutrophil-mediated impairment of ileal microvascular regulation after bypass, but does not improve extravascular mesenteric dysfunction after CPB. (Ann Thorac Surg 2000;69:799 – 807) © 2000 by The Society of Thoracic Surgeons

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attenuate endothelium-dependent relaxation [5], activate neutrophils [6] and induce P-selectin and other adhesion molecules on the endothelial cell surface [7]. Direct and indirect complement-mediated effects, resulting from the deposition of C5b-9, may act synergistically with the toxic effects of activated neutrophils to cause microvascular damage. Mesenteric vasoconstriction and increased vascular permeability, tissue edema, and neutrophil sequestration and activation, may lead to mucosal damage and gut epithelial hyperpermeability. Potent vasopressors, such as norepinephrine and phenylephrine, are often administered after CPB to maintain systemic blood pressure. It is hoped that by maintaining systemic perfusion pressure, blood flow to the gut, kidneys and other visceral organs can be supported. Translocation of bacteria and leakage of endotoxin or other microbial products from the lumen of the ischemic or dysfunctional gut into the systemic circulation might result from these inflammatory mechanisms, and perhaps contribute to much of the morbidity of CPB after cardiac operation involving extracorporeal circulation [8]. In this study, we assessed the physiologic effects of CPB on mesenteric parenchymal and microvascular dysfunction, both during and after CPB. We also examined how the functional and selective inhibition of comple-

esenteric complications occasionally occur after cardiopulmonary bypass (CPB), and mesenteric mucosal ischemia and acidosis have been documented during extracorporeal circulation, despite normal or supranormal flow in the larger mesenteric vessels [1, 2]. These findings suggest that gut injury and inflammation occur during CPB despite normal systemic perfusion. Activation of complement occurs with the initiation of CPB [3] mainly through activation of the alternative cascade, and leads to increased circulating levels of the anaphylatoxins C3a and C5a. These complement fragments activate and are chemotactic for neutrophils, induce cytokine expression, and contribute to systemic inflammation during cardiac operation. In addition to initiating leukocyte-mediated injury during CPB, C5a can have direct vasoconstrictive effects on blood vessels, contributing to further end-organ injury. Furthermore, the terminal membrane attack complex of the complement cascade, C5b-9, may play a major role in mediating systemic inflammatory reactions induced by CPB. The deposition of C5b-9 can be observed on endothelial cells soon after ischemia-reperfusion [4], and can directly

Accepted for publication Aug 30, 1999. Address reprint requests to Dr Sellke, Division of Cardiothoracic Surgery, Beth Israel-Deaconess Medical Center, 110 Francis St, LMOB 2A, Boston, MA 02215; e-mail: [email protected].

© 2000 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

0003-4975/00/$20.00 PII S0003-4975(99)01408-3

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ment fragment C5a with a monoclonal antibody ameliorates mesenteric vascular dysfunction observed after CPB using a clinically relevant in vivo model of extracorporeal circulation.

Material and Methods Animal Preparation Yorkshire pigs (20 to 25 kg) of either sex were premedicated with ketamine (10 mg/kg, IM) and anesthetized with ␣-chloralose and urethane (60 mg/kg and 300 mg/kg IV initially, and then 15 mg/kg and 60 mg/kg every 60 minutes as needed, respectively). Pigs were tracheally intubated and mechanically ventilated (Harvard Apparatus Inc, South Natick, MA). In the base line control group (n ⫽ 6), sternotomy and laparotomy were performed before the pig was heparinized (500 U/kg). A portion of terminal ileum was excised and immediately placed in a cold (4 to 10°C) Krebs’ buffer solution of the following composition (in mmol/L): 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 25.0 NaHCO3, and 11.1 glucose. Another portion of ileum was placed in liquid nitrogen. After induction of anesthesia and tracheal intubation, fluid-filled catheters were introduced into the femoral artery and vein of 12 pigs, for the measurement of peripheral arterial pressure and the infusion of fluorescein isothiocyanate-dextran (FD-4) with an average molecular weight of 4,000 Daltons. Through a midline laparotomy, a 6-mm ultrasonic flow probe (model 6R, Transonics System, Inc, Ithaca, NY) was positioned around the origin of the superior mesenteric artery (SMA) and connected to a flow monitor (model T206, Transonics System, Inc). A 2.5F catheter was placed in the superior mesenteric vein (SMV) through an ileal tributary. During the period of abdominal surgical preparation and recovery, Ringer’s lactate solution (RL) was infused intravenously at 25 mL 䡠 kg⫺1 䡠 h⫺1. Three 15-cm segments of distal ileum were isolated with umbilical ties. A tonometric catheter tipped with a CO2-permeable silastic balloon (Tonometrics Inc, Worcester, MA) was inserted into the distal segment of the ileal lumen through a small antimesenteric enterotomy and secured in place with a purse-string suture. The lumen of the most proximal isolated segments was cannulated with catheters at its proximal and distal ends through small antimesenteric enterotomies, which were rendered watertight with purse-string sutures. The segment was flushed with 500 mL of RL warmed to 37°C, and then perfused continuously with 37°C RL at 60 mL/hour throughout the study. In the middle segment, a specially modified laser-Doppler flow probe (Transonics Systems, Inc) was secured to the antimesenteric mucosal surface and connected to a flow monitor (Model ALF 21, Transonics Systems, Inc). Upon completion of the surgical procedure, the abdominal wall was closed with a running suture, and the pigs were permitted to stabilize for 1 hour. During the period of stabilization a sternotomy was performed, and purse-string sutures were placed in the

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distal ascending aorta and the right atrium. In 6 pigs, anti-C5a monoclonal antibody (C5a MAb, 1.6 mg/kg) was administered intravenously 20 minutes before the onset of CPB, while 6 pigs received saline vehicle only. Pigs were heparinized (500 U/kg) and cannulated through the distal ascending aorta and the right atrium. 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. Blood flow was maintained from 80 to 100 mL 䡠 kg⫺1 䡠 min⫺1 to maintain a mean perfusion pressure of 40 to 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) before commencing CPB, and at 15 minute intervals thereafter. Arterial blood gases were adjusted by ventilatory rate and tidal volume to maintain Pao2 more than 50 mm Hg, PaCO2 more than 30 and less than 45 mm Hg, and pH between 7.35 and 7.45. Pigs were weaned off CPB and then 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 120 minutes of post-CPB perfusion, one portion of the terminal ileum was excised and immediately placed in a cold Krebs’ buffer solution, and another 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 Science and published by the National Institutes of Health (National Institutes of Health publication No. 80 to 23, revised 1987).

Gut Function Measurements The following variables were measured every 30 minutes: SMA flow (QSMA) [mL/min], ileal mucosal flow (Qmuc) [mL/min/100 g]. Blood was also obtained every 30 minutes from the femoral artery and SMV for the measurement of Pao2, PaCO2, Cao2, PSMVO2, PSMVCO2, and CSMVO2. Saline from the tonometer was sampled at the same time, for the measurement of PtonCO2, PtonO2, and pH measurement, using a pH blood gas analyzer (Instrumentation Laboratories, Lexington, MA). PtonCO2 was corrected for incomplete equilibration during the 30-minute sampling period by multiplying PtonCO2 by 1.26, as recommended by the manufacturer (Tonometrics Inc, Worchester, MA). Ileal mucosal pH ([pH]muc) was calculated as follows: [pH]muc ⫽ 6.1 ⫹ log10([HCO3⫺]art/ 1.26 ⫻ PtonCO2 ⫻ 0.03). Ileal oxygen consumption (Vo2) was calculated as follows: 0.01 ⫻ QSMA ⫻ arteriovenous O2 content difference (Cao2 ⫺ CSMVO2). Ileal oxygen delivery (Do2) was calculated as follows: 0.01 ⫻ QSMA ⫻

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Cao2, where Cao2 and CSMVO2 were oxygen content of SMA and SMV blood [mL O2/dL blood]. Oxygen contents were calculated as follows: (Hgb [gm/dL] ⫻ 1.39 ⫻ SO2) ⫹ (Po2 ⫻ 0.00314). Fractional oxyhemoglobin saturation (SaO2 and SSMVO2) was measured with a model 482 CO-oximeter (Instrumentation Laboratories). Mucosal permeability in the proximal segment of the ileum was determined as previously described [9] by measuring the plasma-to-lumen clearance of a hydrophilic macromolecular solute (FD-4) with 14 to 20 Å in diameter. FD-4 in RL at a concentration of 15 mg/mL was infused, as an intravenous bolus, more than 5 minutes (10 mg/kg), followed by continuous infusion (5 mg 䡠 kg⫺1 䡠 h⫺1) for the duration of the study. Beginning at ⫺60 minutes (minus denotes before initiation of CPB), 0.5 mL of arterial blood was sampled every 15 minutes and placed in a CAPIJECT tube (Terumo Medical Corp, Elkton, MD) on ice, in the dark until assayed. For the determination of the plasma FD-4 concentration, the CAPIJECT tubes were centrifuged at 3,000 rpm for 10 minutes at 4°C. The plasma was aspirated and diluted 1:300 in ultrapure distilled water. Fluorescence of FD-4 was measured with a Perkin-Elmer LS-50 fluorescence spectrophotometer (excitation wavelength ⫽ 492 nm, excitation slit width ⫽ 10.0 nm, emission wavelength ⫽ 515 nm, emission slit width ⫽ 10.0 nm, integration time ⫽ 60 seconds). The perfusate exiting from the cannulated ileal segment was collected in a specimen container (Oxford, St. Louis, MO) every 30 minutes, and placed on ice in the dark. The volume of the effluent was recorded and centrifuged at 3,000 rpm for 10 minutes at 4°C, and the fluorescence of a 1 to 3 dilution, in ultrapure distilled water, was determined. Flux of FD-4 was calculated as the product of effluent volume times the fluorescence intensity of the effluent. Clearance of FD-4 was calculated according to the formula: Clearance [␮l/min/cm ileum] ⫽ (Cper ⫻ Q)/(Cpl ⫻ L) ⫻ 1000, where Cper and Cpl represent the fluorescence intensity of the perfusate and plasma, respectively; Q represents the luminal perfusion rate (60 mL/min); and L represents the length of the perfused segment of the ileum (15 cm).

Percent Tissue Water The tissues from the terminal ileum were weighed, incubated at 110°C for 12 hours, and then weighed again. Percent tissue water was calculated as (wet weight-dry weight)/wet weight ⫻ 100.

Microvessel Studies Arterial microvessels (88 to 168 ␮m in internal diameter) were dissected from the ileum using a 10 to 60 ⫻ dissecting microscope (Olympus Optical, Tokyo, Japan). Microvessels were placed in a specially designed microvessel chamber (University of Iowa Medical Instrumentation, Iowa City, IA), cannulated with dual glass micropipettes measuring 40 to 60 ␮m in diameter, and secured with 10 to 0 nylon monofilament suture (Ethicon, Somerville, NJ). Oxygenated (95% O2/5% CO2) Krebs’ buffer solution warmed to 37° C was continuously circulated through the microvessel chamber. The microvessels

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were pressurized to 40 mm Hg in a no-flow state, using a burette manometer filled with Krebs’ buffer solution. With an inverted microscope (⫻ 40 to 200, Olympus CK2, Olympus Optical, Tokyo) connected to a video camera, the vessel image was projected onto a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure internal lumen diameter. Microvessels were allowed to bathe in the organ chamber for at least 30 minutes before an intervention.

Microvessel Study Protocols In all experimental groups, the relaxation responses of the microvessels were examined after precontraction of the microvessels, with the thromboxane A2 analogue U46619, by 30 to 70% of the base line diameter. Base line diameter was defined as the diameter measured within minutes of cannulation and equilibration in the buffer solution. Minimal if any spontaneous tone developed during the equilibration period ( ⬍ 5%). Once the steadystate tone was reached, the relaxation responses of the ileal microvessels to adenosine 5⬘-diphosphate (receptormediated endothelium-dependent vasodilator, ADP) (10⫺9 ⫺ 10⫺4 mol/L), substance P (endothelium-dependent vasodilator, 10⫺12 ⫺ 10⫺7 mol/L), sodium nitroprusside (endothelium-independent vasodilator, SNP) (10⫺9 ⫺ 10⫺4 mol/L), and isoproterenol (␤-adrenoceptor agonist) (10⫺12 ⫺ 10⫺6 mol/L) were examined. In addition, the contraction responses of ileal microvessels to phenylephrine (␣1-adrenoceptor agonist, 10⫺9 ⫺ 10⫺4 mol/L) and to the protein kinase C activator 12-deoxyphorbol 13-isobutyrate 20 acetate (phorbol ester, 10⫺5) were examined. Measurements were made 2 to 3 minutes after the drug administration, except for phorbol ester. In this case, 12-deoxyphorbol 13-isobutyrate 20 acetate was applied to vessels, and contraction over time was measured. All drugs were applied extraluminally. One to four interventions were performed on each vessel. The order of the drug administration was random. Once phorbol ester was applied to a vessel, it was discarded. The microvessels were washed with a Krebs’ buffer solution, and allowed to equilibrate in a drug-free Krebs’ buffer solution for 15 to 30 minutes between interventions. After completion of experiments on a vessel, papaverine (100 ␮mol/L) was added to verify that the initial base line diameter and maximally dilated diameter were similar.

Tissue Myeloperoxidase Activity and Hemolytic Complement Assay Ileal tissue was excised at the end of reperfusion, frozen in liquid nitrogen and stored at ⫺80°C until assayed. Myeloperoxidase (MPO) activity was measured as previously described by Amsterdam and coworkers [10]. Assessment of this assay with isolated porcine neutrophils, demonstrated a linear relationship (r ⫽ 0.92) between MPO and the number of cells such that 1 unit of MPO activity correlated with 2.9 ⫻ 106 neutrophils. The ability of anti-C5a MAb to inhibit formation of the membrane attack complex was assessed by measurement

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of the effect of this MAb on the hemolytic activity (CH50) of porcine serum as previously described by Amsterdam and colleagues [10].

iNOS Protein Content (Western Analysis) Total protein from ileal tissue (stored at ⫺80°C, as for MPO) was obtained by homogenization in a lysis buffer containing 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, and centrifuged at 12,000 g for 10 minutes 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 minutes in Protein Assay Dye Reagent (Bio-Rad, Hercules CA). Total protein (50 ␮g/lane) was fractionated on 8% SDSpolyacrylamide gel, then transferred to Immobilon-p (Millipore, Bedford, MA). The membrane was incubated with 5% nonfat dry milk powder, 0.05% Tween 20 in phosphate buffer saline (PBS) for 2 hours at room temperature to block nonspecific absorption, and then immunoblotted with the monoclonal mouse antiinducible nitric oxide synthase (iNOS) antibody (Transduction Laboratories, Lexington, KY) to amino-acid sequence 961 to 1144 (1/500 [v/v] dilution) for 2 hours. After washing with PBS, the membrane was incubated for 1 hour in 5% milk powder PBS containing 1:3000 diluted goat antimouse IgG conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA). Peroxidase activity was visualized using an enhanced chemiluminescence substrate system (Amersham, Arlington Heights, IL). Densitometry of digitized immunoblot images (ScanJet 4c, Hewlett Packard) was performed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). A polyclonal antibody raised against the structural protein ␤-actin (1:2000 dilution, Santa Cruz Biotechnology, Inc) was used as a control (42 kd) for equal loading, and the optical density ratio of iNOS to that of ␤-actin was used to correct for protein loading.

Measurements of iNOS mRNA by Semiquantitative RT-PCR Approximately 1 g of ileum was snap frozen in liquid nitrogen, and stored at ⫺80°C. The tissue was homogenized in 4 mol/L guanidium isothiocyanate, followed by centrifugation through 5.7 mol/L cesium chloride at 200,000 g for 16 hours. iNOS mRNA was amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR). Sense and antisense primers were designed based on the published human iNOS sequence [11], and on the published porcine eNOS sequence. The sense primer 5⬘-GCCTCGCTCTGGAAAGA-3⬘, corresponding to bases 1425 to 1441, and the antisense primer 5⬘-TCCATGCAGACAACCTT-3⬘ corresponding to bases 1908 to 1924, were used to amplify a 500 base-pair fragment of iNOS [11]. Equal amounts of total RNA were used for RT-PCR from control and experimental groups. For quantification, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified from the same amount of RNA

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to correct for variations in sample mass. The PCR products were loaded in 1.5% agarose gel, scanned, and then densitometry was performed using Image-Quant software.

Drugs Anti-C5a MAb was produced as previously described [12]. FD-4, U46619, isoproterenol, adenosine 5⬘-diphosphate, substance P, and sodium nitroprusside were obtained from Sigma (St. Louis, MO). Phenylephrine and 12-deoxyphorbol 13-isobutyrate 20 acetate were obtained from RBI (Natick, MA). Drugs were dissolved in ultrapure distilled water, and prepared on the day of the study.

Data Analysis The relaxation responses were expressed as the percent relaxation (mean ⫾ standard error of mean, SEM) of the U46619-precontracted diameter of the microvessels. Contraction responses were expressed as % contraction of the base line diameter. Comparisons of dose-response curves between experimental groups were performed by 1-way and 2-ways 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 or functional variables, CH50, MPO activity, and wet/dry between the saline and MAb C5a-treated groups. Statistical significance was taken at p less than 0.05.

Results Mesenteric and Mucosal Perfusion and Ileal Epithelial Permeability Despite a reduction in systemic perfusion pressure during CPB, blood flow in the superior mesenteric artery was not significantly decreased (15% and 11% in saline and C5a Mab groups, respectively). However, clearance of FD-4 increased, and Qmuc and the ratio of Qmuc/QSMA both remained significantly lower than base line during and after CPB (Table 1). Mesenteric oxygen consumption (Vo2) significantly increased (p ⬍ 0.05 vs base line) and, in conjunction with this increase, mesenteric oxygen delivery (Do2) was significantly decreased (p ⬍ 0.05 vs base line) after CPB. [pH]muc decreased dramatically (p ⬍ 0.05 vs base line) after CPB, despite the adjustment of metabolic acidosis for the arterial blood and remained significantly acidotic (p ⬍ 0.05 vs base line) even at 60 minutes of reperfusion. Interestingly, and contrary to our hypothesis, there was no benefit observed with the administration of C5a MAb.

Wet/Dry Ratio Wet/dry ratios were 4.73 ⫾ 0.06, 6.02 ⫾ 0.24, and 6.22 ⫾ 0.09 in the base line control, saline, and C5a MAb groups, respectively. Although a significant increase (p ⬍ 0.05) was noted between control and saline groups, C5a MAb did not reduce the ratio.

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Table 1. Mesenteric (Ileal) Function Before and After CPB and Post-CPB-Perfusion Saline

Clearance of FD-4 Qmuc % Q muc/sma ratio VO2 DO2 [H⫹]muc/[H⫹]a [pH]muc QPA MBP

C5a MAb

BL

30 min CPB

60 min Post

120 min Post

BL

30 min CPB

60 min Post

120 min Post

0.18 ⫾ 0.04 15.9 ⫾ 2.6 3.8 ⫾ 0.6 7.4 ⫾ 0.8 46.4 ⫾ 6.3 1.06 ⫾ 0.11 7.42 ⫾ 0.04 2.60 ⫾ 0.30 89 ⫾ 7

0.44 ⫾ 0.14 9.3 ⫾ 1.0a 3.0 ⫾ 0.3 10.1 ⫾ 1.2 20.0 ⫾ 1.9b 1.74 ⫾ 0.29 7.27 ⫾ 0.08 ... ...

0.64 ⫾ 0.19 10.1 ⫾ 1.8 2.7 ⫾ 0.3a 15.4 ⫾ 1.2b 25.9 ⫾ 3.3a 2.48 ⫾ 0.5a 7.01 ⫾ 0.11b 1.98 ⫾ 0.25a 63 ⫾ 5a

0.49 ⫾ 0.11 10.4 ⫾ 1.9 3.0 ⫾ 0.4 12.3 ⫾ 2.3 22.7 ⫾ 2.6b 1.88 ⫾ 0.37 7.20 ⫾ 0.09 1.75 ⫾ 0.17a 55 ⫾ 5a

0.20 ⫾ 0.02 18.9 ⫾ 3.3 4.1 ⫾ 1.3 7.4 ⫾ 1.3 54.5 ⫾ 7.3 0.92 ⫾ 0.04 7.45 ⫾ 0.04 2.45 ⫾ 0.24 88 ⫾ 4

0.36 ⫾ 0.06 12.7 ⫾ 3.2 2.9 ⫾ 0.9 12.8 ⫾ 1.6a 25.6 ⫾ 2.0b 1.27 ⫾ 0.05b 7.36 ⫾ 0.02 ... ...

0.82 ⫾ 0.21 9.9 ⫾ 1.4a 2.4 ⫾ 0.8 15.8 ⫾ 2.0b 26.7 ⫾ 5.1a 2.17 ⫾ 0.20b 6.97 ⫾ 0.06b 1.88 ⫾ 0.23 61 ⫾ 4a

0.34 ⫾ 0.06a 8.8 ⫾ 0.8a 2.1 ⫾ 0.6a 12.1 ⫾ 3.3 20.7 ⫾ 5.1b 1.92 ⫾ 0.40a 7.15 ⫾ 0.08a 1.71 ⫾ 0.28a 56 ⫾ 6a

a

a

a

a

Data are mean ⫾ SE. BL ⫽ baseline; 30 min CPB ⫽ 30 min after cardiopulmonary bypass; 60 min REP ⫽ 60 min after reperfusion; 120 min REP ⫽ 120 min after MBF ⫽ ileal mucosal reperfusion; Clearance of FD-4 ⫽ clearance of fluorescein isothiocyanate-dextran for the previous 30 min [␮l/min/cm ileum]; blood flow [Qmuc, ml/min/100 g]; % Q ratio ⫽ a ratio of ileal Qmuc to superior mesenteric artery (SMA) blood flow (QSMA) calculated as follows: 100 ⫻ Qmuc/QSMA; VO2 ⫽ ileal oxygen consumption calculated as follows: 0.01 ⫻ QSMA ⫻ (CaO2 ⫺ CSMVO2) [ml O2/min]; DO2 ⫽ ileal oxygen delivery calculated as follows: 0.01 ⫻ QSMA ⫻ CaO2 [ml O2/min]; [H⫹]a ⫽ a ratio of ileal mucosal [H⫹] to femoral artery [H⫹]; [pH]muc ⫽ ileal mucosal b a intracellular pH. QPA ⫽ pulmonary artery blood flow [L/min, total cardiac output], p ⬍ 0.01 vs baseline and p ⬍ 0.05 vs baseline.

Ileal Microvessel Characteristics Ileal microvessels ranged from 88 to 168 ␮m in internal diameter, averaging 133 ⫾ 9 in the control group, and 137 ⫾ 7 and 138 ⫾ 6 ␮m in the saline and C5a MAb groups, respectively. Percent precontraction after application of U46619 was 55% ⫾ 5% in the control group, 58% ⫾ 5% and 54% ⫾ 4% in the saline and C5a MAb groups, respectively. Mean concentrations of U46619 required to obtain these % contractions were (0.7 ⫾ 0.3) ⫻ 10⫺7, (1.9 ⫾ 0.9) ⫻ 10⫺7, and (7.8 ⫾ 2.2) ⫻ 10⫺7 mol/L in the control, saline and C5a MAb groups, respectively.

significantly attenuated with C5a MAb to the level observed in control vessels (p ⬍ 0.05 vs saline group) (Fig 5). The response to phorbol ester was increased after CPB. This enhanced response was not affected by C5a MAb (Fig 6).

MPO Activity MPO activities were 19 ⫾ 9 in the control group, 83 ⫾ 13 (p ⬍ 0.05 vs control and C5a MAb groups) and 41 ⫾ 9 (p ⬍ 0.05 vs control group) units/g protein in the saline and C5a MAb groups, respectively.

Endothelium-Dependent Relaxation The responses to the receptor-mediated endotheliumdependent vasodilator ADP were significantly reduced at the higher concentration in the saline group (p ⬍ 0.05 vs control group) (Fig 1). C5a MAb improved the response to ADP at the higher concentration (p ⬍ 0.05 vs saline group at 10⫺6 mol/L ADP). The responses to substance P at higher concentrations were slightly improved with C5a MAb compared to those in the saline group (p ⬍ 0.01 and p ⬍ 0.05 vs control and C5a MAb group at 10⫺7 and 10⫺8 mol/L substance P, respectively) (Fig 2).

Endothelium-Independent Cyclic GMP and ␤-Adrenergic Relaxations Relaxation responses to SNP were similar in all groups, indicating no alteration in the ability of the vascular smooth muscle to relax through the cyclic GMP pathway (Fig 3). Isoproterenol induced a significant relaxation in control microvessels. The relaxation responses to isoproterenol in the saline and C5a MAb groups were unchanged, compared to the control group (Fig 4).

Responses to ␣1-Adrenergic and PKC Activation Contraction responses to phenylephrine were significantly increased in the saline group (p ⬍ 0.05 vs control group). However, the responses to phenylephrine were

Fig 1. In vitro responses of precontracted ileal microvessels to the endothelium-dependent vasodilator ADP from control pigs, and pigs in the saline and C5a MAb groups. Responses are expressed as % relaxation of U46619-induced precontraction. (ADP ⫽ adenosine 5⬘-diphosphate. † p less than 0.05 versus control and C5a MAb groups at 10⫺6 M ADP.)

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Fig 2. In vitro responses of precontracted ileal microvessels to the endothelium-dependent vasodilator substance P from control pigs and pigs in the saline and C5a MAb groups. Responses are expressed as % relaxation of U46619-induced precontraction. * p less than 0.01 and † p less than 0.05 versus control and C5a MAb groups at 10⫺7 and 10⫺8 M substance P.

Total Hemolytic Complement Activity (CH50) The total hemolytic complement activity was 0.2 ⫾ 0.1 (CH50 units, pre-CPB) and 0.6 ⫾ 0.2 (2 hours post-CPB, p ⬍ 0.05 vs pre-CPB) in the C5a MAb group, compared to 0.1 ⫾ 0.1 and 0.7 ⫾ 0.2 (p ⬍ 0.05 vs pre-CPB), respectively, in the saline group. There was no significant difference

Fig 3. In vitro responses of precontracted ileal microvessels to the endothelium-independent cyclic GMP-mediated vasodilator SNP from control pigs and pigs in the saline and C5a MAb groups. Responses are expressed as % relaxation of U46619-induced precontraction. (SNP ⫽ sodium nitroprusside.)

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Fig 4. In vitro responses of precontracted ileal microvessels to the endothelium-independent cyclic AMP-mediated vasodilator isoproterenol from control pigs and pigs in the saline and C5a MAb groups. Responses are expressed as % relaxation of U46619-induced precontraction.

between the saline and C5a MAb groups at 2 hours post-CPB.

Expression of iNOS in the Ileum iNOS protein was equally expressed in the ileum under base line conditions, and after CPB as assessed by West-

Fig 5. In vitro contraction responses of noncontracted ileal microvessels to phenylephrine from control pigs and pigs in the saline and C5a MAb groups. Responses are expressed as % diameter change of base line diameter. Minus denotes contraction. † p less than 0.05 versus control and C5a MAb groups.

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Fig 8. Expression of iNOS mRNA by reverse transcription polymerase chain reaction (RT-PCR) in the ileum of control pigs and pigs in saline and C5a Mab groups. GAPDH was used to determine equal RT-PCR reaction among groups.

Fig 6. In vitro contraction responses of noncontracted ileal microvessels to the PKC activator phorbol ester (10 ␮mol/L) from control pigs and pigs in the saline and C5a MAb groups. Responses are expressed as % diameter change of base line diameter over time. Minus denotes contraction. † p less than 0.05 versus control and C5a MAb groups.

ern blotting (Fig 7). Similarly, the mRNA levels of iNOS, as assessed by semiquantitative RT-PCR, was not altered by CPB (Fig 8).

Comment The findings of this study are that CPB causes reductions in mucosal blood flow and mucosal pH, and leads to increases in mesenteric edema (vascular permeability), epithelial permeability, microvascular endothelial dysfunction, and mesenteric sequestration of neutrophils. In addition, ileal microvessels are hypercontractile to an ␣1adrenergic agonist and an activator of protein kinase C after CPB. Total complement activity is increased during CPB, likely contributing in large part to the mesenteric

Fig 7. Ileal content of iNOS protein (Western blotting) in control pigs and pigs in saline and C5a MAb groups. B-actin was used to evaluate proper protein loading and transfer.

dysfunction. The functional and selective inhibition of C5a, significantly limited leukocyte-mediated impairment of mesenteric microvascular endothelium-dependent relaxation and the ␣1-adrenergic-mediated hypercontractile response after CPB, but had no effect on the enhanced contractile response to phorbol ester. However, despite its effect on improving vasomotor regulation and significant limitation of neutrophil sequestration, C5a MAb had no effect on the deterioration in mucosal blood flow and acidosis, increased mesenteric edema or increased epithelial permeability after CPB. The failure of C5a MAb to reduce the mesenteric injury may be due to several reasons. First, there may be a differential effect of reduced leukocyte chemotaxis in mesenteric parenchyma and in the mesenteric microcirculation. Secondly, post-CPB mesenteric injury may occur due to complement activation, the generation of the membrane attack complex (C5b-9), increased oxidative stress and oxygen free radical generation, and other mechanisms not directly related to neutrophil activation, adherence, and sequestration. Nitric oxide has been implicated in mediating much of the damaging inflammatory effects of macrophages and cytokines. Indeed, excessive production of NO has been shown to cause intestinal epithelial hyperpermeability, both in vitro in cultured cells [13] and in vivo in rats [14]. iNOS was expressed under base line conditions as previously described by others [15], but expression was not increased after CPB. This finding suggests that the mesenteric injury is not a consequence of increased expression of iNOS, at least within the time constraints of the study. The C5b-9 terminal membrane attack complex has a direct action of cytolytic activity [16] and both C5b-9 [2] and zymosan-activated complement [17] have been shown to impair endothelium-dependent relaxation. The effects of the complement anaphylatoxin C5a include chemotaxis, activation, aggregation, and plugging of leukocytes in the microcirculation [18, 19]. These actions may lead to increased vascular permeability and the release of vasoactive and inflammatory substances such as leukotrienes, thromboxane A2, cytotoxic proteases, and oxygen-derived free radicals. Although C5a itself

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does not attenuate endothelium-dependent relaxation in the porcine vasculature [5], activation of the alternative complement pathway during CPB is relevant to the occurrence of endothelial dysfunction through the above mechanisms. Reduced endothelium-dependent relaxation of the ileal microvessels to ADP and substance P was significantly improved, along with a reduction of MPO activity in the ileum in this model simulating clinical CPB. These findings clearly demonstrate reduced neutrophil infiltration with the administration of C5a MAb. These findings suggest that the C5b-9 terminal membrane attack complex may not be the only cause of ileal endothelial dysfunction during CPB, and a synergistic relationship between the effects of C5a, C5b-9 and other complement fragments and mechanisms of endothelial dysfunction is suggested. Mesenteric ischemia and hypoperfusion led to increased mucosal hyperpermeability and mucosal acidosis. The movement of FD-4 is primarily impeded by the mucosal barrier [20]. Therefore, the clearance of FD-4 is highly correlated with reduction in mucosal blood flow (Qmuc, MBF) and associated intramucosal acidosis. Arteriolar smooth muscle tone largely controls blood flow to the gut [21], and is governed in part by the local oxygen tension [22]. A reduction in MBF and [pH]muc in our study is suggestive of local ischemia in the ileum (Table 1). The decreased oxygen delivery observed in this study may be explained by a combination of local factors, such as edema, altered vascular tone, and by hemodilution. It has been reported [9] that [pH]muc correlates well with a reduction of MBF, and a reduction in MBF and [pH]muc may be associated more with nonpulsatile flow than with pulsatile flow [23]. Furthermore, normothermic CPB is also associated with a reduction in MBF, despite the well-maintained SMA flow. In some pigs, regardless of the optimal maintenance of SMA flow and fluid resuscitation, mucosal hyperpermeability, muscosal hypoperfusion, and tissue edema were increased immediately after commencement of CPB, suggesting an effect of nonpulsatile flow, altered autoregulation, or other neurohumoral effects.

Clinical Implications and Limitations Mesenteric hypoperfusion, edema formation, and increased epithelial permeability may have significant impact on the recovery of patients after cardiac operations utilizing CPB [8]. It was hypothesized that the administration of a monoclonal antibody to C5a would improve these indices of mesenteric function, and possibly provide a method to reduce the morbidity of patients undergoing cardiac operation. However, despite reducing neutrophil infiltration and normalizing vasomotor regulation, MAb C5a had little effect on small bowel dysfunction. Previous studies have found that other complement fragments, most notably C5b-9 (terminal membrane attack complex), may lead to both endothelial dysfunction and cause activation, chemotaxis, and adhesion of neutrophils. MAb C5a had no effect on total complement activation (CH50), and thus did not affect complement activation in general nor decrease production of C5b-9. Therefore, other components of the com-

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plement cascade are still active and may have accounted for the minimal effects of C5a inhibition on indices of mesenteric function and viability. Inhibitors of complement activation should, on a theoretical basis, lessen organ dysfunction during and after CPB [4]. However, there is a controversy on the effectiveness of the treatment with soluble complement receptor-1 and other inhibitors of complement activation on organ dysfunction [24]. Complement activation during CPB does not necessarily account for all of the observed tissue damage. Leukocyte depletion is generally not sufficient to completely abolish mesenteric injury, or even reduce it significantly. Collectively, an inhibition of either complement cascades or fragments may not be enough to fully inhibit systemic inflammatory responses during and after CPB. It may be that the monoclonal antibody to C5a was administered in an insufficient concentration or was not qualitatively effective in limiting neutrophil sequestration. However, we have previously shown that this concentration of C5a MAb inhibits neutrophil chemotaxis and aggregation in vitro, and that the amount of antibody administered was sufficient to attain circulating levels to provide this concentration [12]. Furthermore, a significant although incomplete inhibition of neutrophil sequestration was observed. It should also be noted that a bubble oxygenator was used rather than a membrane oxygenator. While the quantitative effects may have been affected by the type of oxygenator used, similar qualitative effects would likely have been observed with either type oxygenator. The mesenteric vascular effects of CPB may have significant clinical relevance. The hypercontractile response of microvessels to phenylephrine and impaired endothelium-dependent relaxation may contribute to reduced mesenteric perfusion after cardiac operation. This is especially true when vasopressor drugs are administered to maintain perfusion pressure to other vascular beds. Since the response of the skeletal muscle [25] arterioles to phenylephrine is reduced, and the response of the mesenteric arterioles is increased, the postoperative infusion of norepinephrine, phenylephrine, or vasopressin may cause a selective vasoconstriction of mesenteric vessels and lead to the development of acidosis and transmural mesenteric ischemia. Indeed, postoperative acidosis, in the absence of an obvious cause after cardiac operation, is not uncommon. In summary, CPB is associated with mucosal hypoperfusion and acidosis, and altered microvascular regulation. The selective and functional inhibition of C5a limits the impairment of endothelium-dependent relaxation, and improved the hypercontractile vascular state in the ileum, likely due to reduced leukocyte chemotaxis, while not inhibiting complement activation. Despite inhibition of C5a and preservation of vascular regulation, C5a MAb has no effect on functional improvement of the ileum during and after CPB. The authors acknowledge the excellent technical assistance of Jennifer M. Lee, Alvin Franklin, Hai Bin Dai, Hailong Wang, and Jianyi Li. This study was supported by NIH grants HL46716

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(Frank W. Sellke), HL52886 (Gregory L. Stahl), HL56086 (Gregory L. Stahl), and GM37631 (Mitchell P. Fink).

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