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Ann Thorac Surg 1998;65:973-977
© 1998 The Society of Thoracic Surgeons

Soluble Complement Receptor Type I Limits Damage During Revascularization of Ischemic Myocardium

^a Department of Cardiothoracic Surgery, Boston Medical Center, Boston, Massachusetts, USA
^b Boston University School of Medicine, Boston, Massachusetts, USA

Accepted for publication October 13, 1997.

Address reprint requests to Dr Lazar, Department of Cardiothoracic Surgery, Boston Medical Center, 88 E Newton St, Suite B404, Boston, MA 02118

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. This study was undertaken to determine whether suppression of complement activation with soluble human complement receptor type I reduces myocardial damage during the revascularization of ischemic myocardium.

Methods. In 20 pigs, the second and third diagonal coronary arteries were occluded for 90 minutes, followed by 45 minutes of cardioplegic arrest and 180 minutes of reperfusion. In 10 pigs, soluble human complement receptor type I (10 mg/kg) was infused over 30 minutes before the period of coronary occlusion; 10 other pigs received no soluble human complement receptor type I. Complement activation was measured by total hemolytic complement activity (expressed as a percentage of preischemic values). Ischemic damage was assessed by changes in myocardial tissue pH, wall motion scores (range, 4 = normal to -1 = dyskinesia), and infarct size (area of necrosis versus area at risk).

Results. After 180 minutes of reperfusion, hearts treated with soluble human complement receptor type I had significantly less complement activation than nontreated hearts (1.1% ± 0.09% versus 7.8% ± 0.04%, respectively; p < 0.002), less myocardial acidosis (-0.41 ± 0.03 versus -0.72 ± 0.03, respectively; p < 0.0001), higher wall motion scores (3.1 ± 0.09 versus 1.67 ± 0.16, respectively; p < 0.0001), and smaller infarct size (24.6% ± 2.0% versus 41% ± 1.3%, respectively; p < 0.0001).

Conclusions. Complement inhibition with soluble human complement receptor type I significantly limits ischemic damage during the revascularization of acutely ischemic myocardium.

	Introduction

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There is growing evidence to suggest that the inflammatory response that results from complement activation during cardiopulmonary bypass (CPB) contributes to postoperative myocardial dysfunction and poor clinical outcomes [1–3]. Soluble human complement receptor type I (sCR-I) is a recombinant form of human complement receptor that is a potent inhibitor of complement activation [4]. The administration of sCR-I has been shown to reduce infarct size in a rat model [4–6], to limit pulmonary dysfunction after CPB in the pig [7], and to delay hyperacute rejection of rat cardiac allografts in presensitized recipients [8]. These beneficial effects of sCR-I suggest that it might be useful in limiting ischemic damage during surgical revascularization for unstable coronary syndromes. This experimental study was undertaken to determine whether inhibiting complement activation with sCR-I would reduce myocardial dysfunction during the revascularization of acutely ischemic myocardium in a porcine model.

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Randomization
Twenty-three pigs were entered into the study. The animals were randomized to receive sCR-I or no sCR-I on an alternating basis. Three animals were excluded from the study, 2 because of pneumonia and 1 because of pericarditis.

Preparation
Twenty adult pigs (34 to 38 kg) were premedicated with intramuscular ketamine (15 mg/kg) and xylazine (0.5 mg/kg), anesthetized with -chloralose (75 mg/kg), and placed on positive-pressure endotracheal ventilation. After a median sternotomy, catheters were placed into the femoral artery and vein for monitoring mean aortic pressure and administering fluids. The azygos vein was ligated to prevent retrograde flow from the coronary sinus during the administration of retrograde cardioplegia. After systemic heparinization (3 mg/kg), the second and third diagonal branches just distal to the takeoff of the left anterior descending artery were occluded with snares for 90 minutes. Intravenous lidocaine was used to treat ventricular arrhythmias.

After the 90-minute period of coronary occlusion, all the animals were placed on CPB (Sarnes Membrane Oxygenator; Sarnes, Inc, Ann Arbor, MI), with a 17F cannula in the femoral artery and a 36F venous return cannula in the right atrium. A 24F cannula was inserted into the left atrium so that volume could be infused to vary the left ventricular end-diastolic pressure. Mean aortic pressure ranged from 65 to 75 mm Hg and pump flow was kept at 80 mL · kg^-1 · min^-1. The hematocrit ranged from 25% to 28%, the pH was maintained between 7.37 and 7.42, and the activated clotting time was 400 seconds or greater.

After the institution of full CPB, all hearts underwent 45 minutes of multidose, antegrade/retrograde, cold blood cardioplegic arrest (potassium = 25 mEq/L, hematocrit = 20, pH = 7.6, temperature = 4°C) supplemented with topical hypothermia. An initial arresting dose (10 mL/kg) was followed by additional doses (5 mL/kg) every 20 minutes. Half of each dose was given antegrade through a catheter inserted into the ascending aorta and the other half through a catheter positioned into the coronary sinus using a pursestring suture in the right atrium (DLP, Inc, Grand Rapids, MI). After the aortic cross-clamp was removed, the coronary snares were released and all hearts were reperfused for 180 minutes on CPB at 37°C.

Treatment groups
In 10 pigs, sCR-I (10 mg/kg; T Cell Sciences, Needham, MA) was infused intravenously over 30 minutes before the period of coronary occlusion. Ten other pigs received no sCR-I but were given a comparable saline solution infusion.

Measurements and statistical analyses
Electrocardiographic leads were placed to measure heart rate and to monitor electrical activity during cardioplegic arrest. A piezoelectric microtip catheter pressure transducer (Millar Instruments, Houston, TX) was inserted into the apex of the left ventricle to measure left ventricular end-diastolic pressure. Systemic body temperature was measured using a rectal temperature probe (Yellow Springs Instruments, Yellow Springs, CO).

Total hemolytic complement activity was determined by a modification of the method of Mayer [9] and was measured during preischemia, 90 minutes after coronary occlusion, and at 60 and 180 minutes during reperfusion. All complement titers were expressed as a percentage of the preischemic values. Previous in vitro studies have demonstrated that human sCR-I is able to completely inhibit activation of both the classic and alternative pathways of porcine complement [7].

Myocardial tissue pH was measured with a pH probe (Khuri Tissue Ischemia Monitor; Vascular Technology, Inc, North Chelmsford, MA) and was standardized according to myocardial temperature as described previously [10]. The pH values were expressed as the change in pH from preischemic values and were recorded for each experiment and then averaged for all experiments in the treated and nontreated groups.

Echocardiographic short- and long-axis sections were used to determine wall motion changes in the area of risk using techniques previously described [10]. A numeral score was used (4 = normal, 3 = mild hypokinesis, 2 = moderate hypokinesis, 1 = severe hypokinesis, 0 = akinesis, -1 = dyskinesia) to indicate the degree of wall motion abnormalities. These scores were averaged for the periods of coronary occlusion and reperfusion for both experimental groups in a blinded fashion by an experienced echocardiographer.

The areas of risk and necrosis were determined by histochemical staining techniques using triphenyltetrazolium chloride (Sigma Chemical Co, St. Louis, MO) as described in our previous study [10]. Stained myocardial slices were plenimetered to obtain the area of risk compared with the total left ventricular mass and the percent area of infarct in that area of risk.

All values represent the mean plus or minus the standard error. Differences in measurements between the treated and nontreated groups and across time were assessed using analysis of variance techniques. In addition, a nonpaired Student’s t test was used to compare the area of necrosis versus the area of risk between the two groups. Data were considered significant at p values less than 0.05. All animals received humane care in compliance with 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 Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
During the 90-minute period of coronary occlusion before cardioplegic arrest, mean aortic pressure (69 ± 3 mm Hg with sCR-I versus 70 ± 2 mm Hg without sCR-I), heart rate (102 ± 3 beats/min with sCR-I versus 100 ± 4 beats/min without sCR-I), and left ventricular end-diastolic pressure (4.0 ± 0.8 mm Hg with sCR-I versus 5.4 ± 1.1 mm Hg without sCR-I) were similar in both groups.

Inhibition of complement activation
Total hemolytic complement activity is shown in Figure 1. After 90 minutes of coronary occlusion, there was significant consumption of complement in animals that did not receive sCR-I (37.0% ± 4.2% compared with preischemic values; p < 0.0001). This continued throughout the period of reperfusion. Plasma from animals that did receive sCR-I showed virtually no hemolytic activity after 90 minutes of coronary occlusion (2.2% ± 1.3%; p < 0.0001 compared with preischemic values and p < 0.0001 compared with animals that did not receive sCR-I). Total hemolytic complement activity in animals that received sCR-I was less than 2% of preischemic measurements during CPB, indicating virtually complete complement inhibition.

View larger version (16K): [in this window] [in a new window]   Fig 1. After 90 minutes of coronary occlusion, plasma from animals treated with soluble CR-I (sCR 1) had virtually no hemolytic complement activity. Total hemolytic complement activity remained less than 2% of preischemic values during reperfusion on cardiopulmonary bypass, indicating almost complete complement inhibition. Data are expressed as a percentage of preischemic values.

View larger version (14K): [in this window] [in a new window]   Fig 2. Hearts treated with soluble CR-I (sCR 1) had significantly less tissue acidosis after 90 minutes of coronary occlusion and 180 minutes of reperfusion. (pH = change in pH.)

View larger version (14K): [in this window] [in a new window]   Fig 3. Wall motion scores were significantly higher in hearts treated with soluble CR-I (sCR 1) after coronary occlusion and reperfusion.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Activation of the complement system during CPB may result in myocardial dysfunction and contribute to postoperative complications. Seghaye and co-workers [3] noted a relation between complement activation and postoperative multisystem organ failure in children after CPB. Hennein and colleagues [11] found that elevation of the proinflammatory cytokines interleukin-6 and interleukin-8 after CPB correlated with new wall motion abnormalities in patients undergoing coronary artery bypass grafting. Interleukin-8 has been shown to play an important role in the regulation of neutrophilic enzyme release and transendothelial migration, and it has been implicated as a major contributor to the post-CPB capillary leak syndrome [12, 13].

Previous attempts to modulate complement activation during CPB have included the use of leukocyte-depleting filters and heparin-bonded circuits. Because complement activation serves as the stimulus for neutrophil-mediated myocardial injury, depleting the blood of neutrophils during CPB and reperfusion may limit ischemic damage. Our own experimental study using a porcine model of acute coronary occlusion and reperfusion on CPB showed that leukocyte-depleting filters placed in the CPB circuit significantly reduced ischemic myocardial damage [14]. Heparin-bonded CPB circuits also have been shown to lower complement levels, decrease neutrophil activation, and reduce serum levels of interleukin-6 and interleukin-8 [1, 2, 15]. In our porcine model of acute coronary occlusion and reperfusion, animals treated with heparin-bonded circuits had the best preservation of wall motion scores, the least tissue acidosis, the smallest increase in lung edema, and the smallest infarct size [16]. In a prospective clinical study involving 234 patients undergoing coronary artery bypass grafting, patients who were treated with heparin-bonded circuits had a lower incidence of myocardial infarction, less need for inotropic support, a lower incidence of prolonged ventilation, and fewer postoperative complications [17]. These studies lend further support to the premise that limiting the inflammatory response after myocardial ischemia and CPB decreases myocardial dysfunction and lowers the incidence of postoperative complications.

Another approach to limiting complement-mediated injury is to "decomplement" the plasma before the induction of myocardial ischemia. Experimental studies showed that depletion of the complement system with cobra venom factor inhibited the deposition of C3 in the ischemic myocardium, attenuated the infiltration of neutrophils, and reduced myocardial infarct size [18]. Compared with cobra venom factor, sCR-I inhibits the complement system without causing the generation of biologically active complement products. Nevertheless, sCR-I cannot participate extensively in the regulation of complement activation because it is found primarily on peripheral blood cells and is not distributed widely among different cell types.

The recombinant soluble form of CR-I retains the inhibitory properties of CR-I and inhibits both the classic and alternative pathways at concentrations 100 times lower than those of other endogenous soluble complement-deactivating proteins. Because it lacks the transmembrane and cytoplasmic domains of CR-I, sCR-I is free to exert its protective effects in a soluble form. Soluble CR-I exerts its biologic actions by binding C3b and C4b to distinct sites, displacing the catalytic subunits from C3 and C5 convertases, and acting as a cofactor in promoting the degradation of C3b and C4b by factor I [4]. In addition to inhibiting complement activation by both the classic and alternative pathways, it indirectly inhibits membrane attack complex, proteins, and the generation of interleukin-8 [19]. Membrane attack complexes are deposited in areas of infarcted and ischemic myocardium. They result in cell lysis by promoting neutrophil adhesion to endothelial cells and by directly attacking cellular membranes.

Soluble CR-I has been shown to decrease myocardial reperfusion damage in several experimental studies. Using a rat model with 30 minutes of coronary occlusion followed by 24 hours of reperfusion, Smith and co-workers [5] found that sCR-I given 5 minutes before coronary occlusion significantly decreased infarct size and significantly reduced the accumulation of neutrophils by reducing myeloperoxidase activity. However, when sCR-I was given only 5 minutes before reperfusion, the decrease in infarct size was not as significant. In a similar model, Weisman and associates [4] found that sCR-I given immediately before 35 minutes of coronary occlusion in the rat significantly decreased infarct size. Further, immunoperoxidase staining showed virtually no membrane attack complex deposition and a significant decrease in leukocytes in the periinfarct zone.

In another isolated rat model with 20 minutes of global ischemia, Shandelya and colleagues [6] showed that sCR-I significantly improved the recovery of postischemic contractile function and prevented the membrane attack complex deposition seen on the surface of small arterioles and capillaries in nontreated hearts. Although sCR-I did not prevent leukocyte adhesion in this model, it decreased complement deposition, which is associated with neutrophil activation and with the generation of oxygen free radicals that result in tissue destruction. Homeister and co-workers [20] used an isolated perfused rabbit heart to determine whether sCR-I would provide protection against tissue injury resulting from exposure of the myocardium to normal human plasma. Ultrastructural examination of tissue from hearts exposed to 6% normal human plasma showed evidence of myocyte injury, with mitochondrial swelling and destruction of the vascular endothelium. These changes were prevented by pretreating the animals with sCR-I.

Our results in a porcine model simulating urgent coronary artery bypass grafting are similar to those of previous experimental studies. Pretreatment with sCR-I resulted in less myocardial tissue acidosis, better preservation of wall motion, and reduced infarct size. Because specific antibodies to measure C3a and C5a are not commercially available for the pig, we were not able to measure directly complement activation during CPB. However, plasma from animals that were treated with sCR-I showed virtually no hemolytic activity, suggesting that complement was actively inhibited. The low level of complement activity in the animals that were not treated with sCR-I most likely represents serum complement depletion resulting from increased deposition in ischemic and infarcted myocardial tissues, as reported previously [4, 20].

The results of this experimental study provide further evidence that interventions that decrease the inflammatory response during myocardial ischemia and CPB limit postoperative myocardial dysfunction. In clinical practice, it is not possible to treat patients with sCR-I before an infarct occurs. Nevertheless, pretreatment of patients who are undergoing coronary artery bypass grafting for unstable coronary symptoms with sCR-I just before the institution of CPB will inhibit complement activation significantly and may decrease further myocardial damage. Clinical studies are being designed to determine whether the favorable effects of sCR-I seen in this experimental model will result in lower morbidity and mortality and shorter hospital stays in patients with unstable angina who are undergoing surgical revascularization on CPB.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
The consultation of Diane Lancaster, PhD, who performed all statistical analyses for this article, is gratefully acknowledged. The secretarial support of Ellie LaBombard is greatly appreciated. Funded in part by a grant from T Cell Sciences, Needham, MA.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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