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Ann Thorac Surg 2007;83:1477-1483
© 2007 The Society of Thoracic Surgeons
a Department of Surgery, Kings College Hospital, London, England
b Department of Surgery, Duke University Medical Center, Durham, North Carolina
c Department of Pediatrics, Duke University Medical Center, Durham, North Carolina
Accepted for publication October 20, 2006.
) = time constant of isovolumic relaxation
* Address correspondence to Dr Jaggers, Pediatric Cardiothoracic Surgery, Duke University Medical Center, DUMC 3474, Durham, NC 27710 (Email: jagge003{at}mc.duke.edu).
Presented at the Forty-second Annual Meeting of The Society of Thoracic Surgeons, Chicago, IL, Jan 30–Feb 1, 2006.
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Abstract |
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Methods: Fifty-two neonatal pigs were randomly assigned to receive 0 IU (n = 22), 500 IU (n = 15), 1,000 IU (n = 8), or 1,500 IU (n = 7) of C1-inh. Doses were delivered 5 minutes before starting 90 minutes of normothermic CPB. Pulmonary and cardiovascular measures were taken before and 5, 30, and 60 minutes after CPB.
Results: Five animals did not survive CPB. The C1-inh concentration post-CPB increased monotonically with increasing dose (p < 0.001). Weight gain was significantly less in the 1,500 IU group (0.24 ± 0.10 kg versus 0.38 ± 0.09 kg, p = 0.001). Dynamic compliance increased with C1-inh dose from 0 to 500 IU by 23% ± 4% (p < 0.001), but the increase leveled off at the higher doses. Alveolar-arterial O2 gradient decreased with C1-inh dose (p = 0.009). Time derivative of left ventricular pressure (dP/dtmax) increased significantly with increasing dose (p = 0.016). At the highest dose of C1-inh, the time constant of isovolumic relaxation was increased (p = 0.018).
Conclusions: The C1-inh supplementation results in improved pulmonary and systolic cardiac function in a model of neonatal CPB. The negative effect on diastolic function requires further investigation.
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Introduction |
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We hypothesize that the greater hemodilution that occurs with CPB in infants and neonates may affect the balance of the regulatory mechanisms that control host inflammatory response. Based on this hypothesis, we tested whether C1-inh supplementation before CPB would ameliorate post-CPB decrease in pulmonary and cardiac function in a neonatal model of CPB.
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Patients and Methods |
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Surgical Procedure
The neonatal pig was anesthetized (20 mg/kg ketamine and 1 mg/kg acepromazine intramuscularly) and mechanically ventilated (Sechrist Infant Ventilator, model IV-100B; Sechrist Industries, Anaheim, CA). A femoral arterial line and nasopharyngeal temperature probe (YSI-400; Yellow Springs Instrument Co, Dayton, OH) were placed. Analgesia was maintained with fentanyl, 100 µg/kg initial bolus and 50 µg · kg–1
· hr–1 continuous infusions. The ventilator provided a positive inspiratory pressure of 25 mm Hg and positive end-expiratory pressure of 3 mm Hg. A pneumotachometer (System 2600 Pediatric Pulmonary Cart; SensorMedics, Conshohocken, PA) was placed in the ventilation circuit. Respiratory rate and inspired oxygen fraction were titrated to maintain an arterial PCO
2 of 35 to 45 mm Hg and PO
2 of 100 to 250 mm Hg. Sodium bicarbonate (8.5%, 10 to 15 mL) was used to maintain a base excess between –3 and +3 mmol/L.
After a sternotomy, umbilical tapes were placed around the superior and the inferior vena cava, and a 10-mm ultrasonic transit-time flow probe (Transonic Systems, Ithaca, New York) was placed around the main pulmonary artery. Micromanometer pressure catheters (3F; Millar Instruments, Houston, Texas) were inserted into the left ventricle (LV), the pulmonary artery at the level of the ultrasonic flow probe, and the left atrium. Two ultrasonic dimension transducers were sewn to the LV epicardium to measure LV base-to-apex major diameter and anterior-to-posterior minor diameter.
Human C1-inh Inhibits Pig C1
We assessed whether human C1-inh inhibits pig C1. Partially purified pig C1 was prepared by euglobulin precipitation followed by FPLC size exclusion chromatography. Pig C1 was able to cleave highly purified human C4. This ability was inhibited in a dose-responsive fashion by purified human C1-inh. In control experiments, it was shown that C1-inh had no ability to cleave C4 directly, and bovine serum albumin (CalBiochem, La Jolla, CA) was unable to inhibit the C4 cleaving activity of pig C1. The results of those experiments demonstrate that human C1-inh used in this study is highly effective in inhibiting pig C1.
Human C1-inh Assay
A sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify C1-inh in pig serum. Plasma levels of endogenous porcine C1-inh could not be determined because no assay for porcine C1-inh was available. Sheep anti-human C1-inh (immunoglobulin G fraction; Binding Site, San Diego, California) was diluted to 1:1,500 in 0.1 mol/L, pH 9.6 carbonate-bicarbonate buffer. The ELISA plate was coated with 100 µL of this solution per well and incubated at 4°C overnight. After washing three times with phosphate-buffered saline (PBS)-Tween, 200 µL of 3% bovine serum albumin in PBS was added to each well and incubated for 1 hour at room temperature. The plate was then washed three times with PBS-Tween. Then 100 µL of diluted pig serum (1:1,000, 1:2000, and 1:4000) was added in duplicate; and 100 µL of C1-inh standards (0.006, 0.032, 0.16, 0.8, 4 and 20 µg/mL) for the standard curve was included in each plate and incubated at room temperature for 1 hour, then washed three times with PBS-Tween. Next, 100 µL of 1:2000 diluted peroxidase labeled sheep anti-human C1-inh was added to each well and incubated for 1 hour at room temperature. After washing again, 100 µL of o-phenylenediamine dihydrochloride peroxidase substrate (Sigma) was added to each well and incubated for 15 minutes at room temperature. Optical density at 405 nm was measured in an ELISA reader. The average C1-inh concentration was calculated from the C1-inh standard curve.
Cardiopulmonary Bypass and C1 Inhibitor Dosing
The C1-inh was given intravenously 5 minutes before CPB. Animals were given heparin (500 IU/kg), and arterial and venous cannulas were placed in the ascending aorta and right atrium. The CPB circuit consisted of a Stockert Shiley roller pump (model 10-10-00; Shiley, Cheshire, CT), a Medtronic Minimax Plus oxygenator (Medtronic, Minneapolis, MN), and a Bio-Cal 370 heat exchanger to maintain piglet temperature at 37°C. The pump was primed with lactated Ringer’s solution and fresh donor pig blood to maintain a circuit hematocrit of 23% to 25%. Cardiopulmonary bypass flow rate was 100 mL · kg–1 · min–1 and mean systemic arterial pressure was maintained at 40 mm Hg. Pigs were maintained on CPB for 90 minutes, then separated from CPB without using inotropic agents. Heparin was not reversed with protamine.
Data Acquisition
Before administration of C1-inh and after CPB, arterial blood gases were stabilized within the above limits. Blood samples were analyzed using a GEM Premier Plus blood gas analyzer (IL Sensor Systems, Ann Arbor, Michigan) to measure PO
2, PCO
2, pH, calcium, and base excess and to derive arteriolar-alveolar oxygen gradient (A-a O2 gradient). Recorded waveforms included LV pressure, left atrial pressure, pulmonary artery pressure, right ventricular output, major and minor LV diameters, and systemic arterial pressure. Nasopharyngeal temperature, dynamic pulmonary compliance, and mean airway resistance were recorded. Data were collected before CPB and at 5, 30, and 60 minutes after CPB. Eight-second data samples were recorded at 500 Hz for all steady-state measurements, and 12-second data samples were recorded at 200 Hz during vena cava occlusion to vary ventricular preload for preload recruitable stroke work (PRSW) calculations. Airway pressure, digitized at 500 or 200 Hz, was stored on a personal computer. Waveform data were filtered using a 50-Hz low pass filter.
Data Analysis
Data were acquired and analyzed with software developed in house. Left ventricular end-diastolic volume was obtained from the sonomicrometric data using an ellipsoid model [4, 5]. Preload recruitable stroke work was calculated as the slope of the LV end-diastolic stroke volume-work relation. The time constant of LV pressure decay during isovolumic relaxation () was determined by fitting a single exponential function to the pressure waveform, beginning at the minimum of the first derivative of LV pressure, by nonlinear regression [6, 7]. Dynamic lung compliance was measured using a Cosmo-Plus respiratory profile monitor (Novametrix, Wallingford, Connecticut).
Statistical Analysis
Values are given as mean ± SE unless stated otherwise. We analyzed the response to C1-inh as a function of dose using linear regression. Where the response versus dose was measured as a function of time post-CPB, we fit the data using linear mixed effects models [8]. We corrected for the pre-CPB values of the response variables (Table 1) by using them as covariates. When controlling for heart rate and instantaneous pressure, we used those variables as covariates. The logarithmic transformation was used for C1-inh plasma concentration, heart rate, dynamic compliance, dP/dtmax, PRSW, and tau () in order to satisfy the statistical assumptions. For comparing the effects of the three C1-inh doses relative to the group receiving no C1-inh (Table 2), we refitted the model using C1-inh dose as a categorical rather than numeric variable. The R [9] and Nonlinear Mixed Effects Model package [8] were used for statistical analysis.
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Results |
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Human C1 Inhibitor
Plasma levels of human C1-inh in animals receiving 500 IU at 5, 30, and 60 minutes were 195 ± 22, 152 ± 18, and 141 ± 17 mg/mL, respectively; in animals receiving 1,000 IU, 689 ± 145, 522 ± 103, and 450 ± 85 mg/mL; in animals receiving 1,500 IU, 922 ± 311, 696 ± 189, and 521 ± 54 mg/mL. Plasma levels of human C1-inh increased significantly by 570 ± 120 mg/mL per 1,000 IU C1-inh dose administered (p < 0.001), and fell with time post-CPB by 190 ± 60 mg/mL per hour (p < 0.001).
Total Body Weight
Net body weight gain post-CPB at 0, 500, 1,000, and 1,500 IU C1-inh was 380 ± 30, 330 ± 10, 380 ± 50, and 140 ± 50 g respectively. Weight gain was significantly less at 1,500 IU than at lower doses (p = 0.001); C1-inh doses 1,000 IU and lower had no significant effect on weight gain (p > 0.49).
Respiratory Function
Alveolar-arterial O2 gradient
The A-a O2 gradient decreased linearly with C1-inh dose (Table 2, p = 0.009) but did not change significantly with time post-CPB.
Dynamic lung compliance
Compliance increased significantly with C1-inh dose from 0 to 500 IU by 23% ± 4% (p < 0.001) but the increase leveled off at the higher doses: from 500 to 1,000 IU dynamic compliance increased by 7% ± 5% (p = 0.21) with virtually no further increase at 1,500 IU (p = 0.83).
Cardiac Function
Heart rate
Heart rate fell with C1-inh dose (p = 0.01) but increased slightly as a function of time (p = 0.03) post-CPB (Table 2).
Maximum rate of pressure development
The LV dP/dtmax increased significantly with increasing dose of C1-inh (p = 0.016). The increase in dP/dtmax with C1-inh remained significant (p = 0.004) when controlled for heart rate, and when adjusted for instantaneous LV pressure (p = 0.008) [10]. There was no significant effect of time post-CPB on dP/dtmax.
Preload recruitable stroke work
We found no significant effect of C1-inh or of time post-CPB on PRSW. Controlling for differences in heart rate did not alter the conclusions.
Time constant of isovolumic relaxation
Complement factor 1 esterase inhibitor did not affect significantly at the 500 and 1,000 IU doses (p > 0.8). However, at 1,500 IU, increased by 58% ± 20% (p < 0.001). After controlling for heart rate, the increase in at 1,500 IU was 31% ± 14% (p = 0.018). The change in with time post-CPB was not significant (Table 2).
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Comment |
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C1 Esterase Inhibitor
Complement factor 1 esterase inhibitor is a serum alpha-2 globulin molecule and a member of the serine family of protease inhibitors. It inhibits both the classic and the alternative complement activation pathway. Complement factor 1 esterase inhibitor binds reversibly to proenzymatic C1r and C1s within intact C1 to prevent their auto-activation or by binding to activated C1r and C1s to dissociate them from C1q. By inhibiting the formation of the C1-esterase complex (C1qC1rC1s), C1-inh prevents the conversion of C4 to C4b and C2 to C2a, thereby preventing formation of C3-convertase (C4bC2a). Complement factor 1 esterase inhibitor can inhibit the alternative complement pathway by binding to C3b and preventing factor B binding to C3b. That prevents the formation of alternative pathway C3-convertase (C3bBb). Complement factor 1 esterase inhibitor also is a major inhibitor of the contact system. The light chain of kallikrein reacts with the C1-inh to form a stoichiometric complex, resulting in loss of proteolytic and amidolytic activity of kallikrein [12]. Complement factor 1 esterase inhibitor is the major inhibitor of factor XIIa, another serine proteinase [13]. It also inhibits both t-PA and u-PA to prevent the formation of plasmin and ultimately decrease fibrinolysis; and it inhibits the intrinsic pathway of coagulation, an essential step in the propagation of thrombin generation.
C1 Inhibitor: Experimental and Clinical Studies
Complement factor 1 esterase inhibitor has been shown to improve right ventricle function in a pig transplant model and to reduce the size of tissue injury in a model of coronary artery occlusion and reperfusion [14, 15]. This inhibitor has also been shown to reduce neuronal injury in a model of middle cerebral artery occlusion and reperfusion and reduces reperfusion injury after experimental lung transplant [16, 17]. These studies support the importance of C1-inh in the pathophysiologic processes associated inflammation and ischemia reperfusion injury.
Clinically, C1-inh is used to treat patients with hereditary angioedema. The success of this therapy and the apparent benign nature of the molecule have suggested its use in other settings in which complement and contact activation play a role in the pathophysiology of the disease. Complement factor 1 esterase inhibitor has also been used to treat the capillary leak syndrome that sometimes complicates bone marrow transplant and also to treat reperfusion injury in patients requiring emergency coronary artery bypass grafting after failed percutaneous angioplasty [18, 19].
C1 Inhibitor in the Neonate and Infant
Complement factor 1 esterase inhibitor levels decrease with CPB in the neonate and infant [3]. Stiller and colleagues [3] examined C1-inh levels in such patients. They found that after CPB, generalized edema, pulmonary edema, and weight gain were greater in infants with lower C1-inh levels. At 30 minutes post-CPB, the infants demonstrating these effects had C1-inh activity levels that were 51% of controls compared with 80% of controls in those who did not demonstrate capillary leak syndrome. In addition, the prekallikrein activity fell further in patients with capillary leaks than in patients without (11% versus 25% at 30 minutes post-CPB and 21% versus 44% at 24 hours post-CPB). The fall in prekallikrein activity is consistent with an increase in the plasma levels of kallikrein. In the same study, factor XIIa increased immediately after CPB. In another study of simulated CPB, there was significant increase in kallikrein-C1-inh complex [20]. The C1-C1-inh complex formation also suggests that factor XII activation occurred.
More recently, in a randomized double-blinded study of 24 neonates undergoing arterial switch for transposition of the great arteries, patients were assigned to either placebo or C1-inh (100 IU/kg as a single dose 30 minutes before CPB) [21]. All 24 patients had an uneventful clinical course. Tassani and coworkers [21] found that mean arterial pressure and pulmonary oxygenation after CPB were superior in patients who received C1-inh. Weight gain on post-CPB days 1 and 4 was significantly less in patients who received C1-inh. Although no influence on levels of C3a and coagulation factors was found, the concentration of IL-6 was significantly lower in those patients. These findings support the need to examine more directly the potential positive and negative effects of C1-inh supplementation on cardiac and pulmonary function in the neonate exposed to CPB.
C1 Inhibitor Effects in a Neonatal Piglet CPB Model
In the current study, we evaluated the effects of C1-inh on cardiac and pulmonary function of piglets in a controlled model of neonatal CPB. The dose chosen ranged from a relatively low dose (about 200 IU/kg) to a relatively high dose (about 600 IU/kg). We chose a model of pure normothermic CPB without cardiac ischemia to try to focus on the effects of CPB and avoid both significant ischemia reperfusion injury and the confounding factors of inotrope therapy present, of necessity, in the clinical study above. In both our previous [22] and current experimental study, the lungs appeared to be very sensitive to the inflammatory effects of CPB. In our study, the primary benefit of C1-inh supplementation appears to be protection of pulmonary function in animals receiving C1-inh supplementation with improved A-a O2 gradient and dynamic pulmonary compliance. We also demonstrated significant improvement in the capillary leak phenomenon by the decrease in total body weight gain at the 600 IU/kg dose of C1-inh supplementation. These beneficial effects may be related to C1-inh inhibition of kallikrein activity and subsequent decreased bradykinin production. Complement factor 1 esterase inhibitor also resulted in a significant improvement in ventricular systolic function as measured by dP/dtmax. On the other hand, our study revealed a possible deleterious effect of the 1,500 IU dose of C1-inh on . An interesting study for the future would be to investigate the potential beneficial effects of C1-inh on a myocardial ischemia reperfusion injury associated with CPB.
In summary, this experimental study demonstrated that C1-inh supplementation before CPB improves pulmonary mechanics and oxygenation in a model of neonatal CPB. We also demonstrate that systolic function may be improved, but that, at very high does of C1-inh, diastolic function may be decreased. Further investigation into the potential beneficial effects of C1-inh is warranted.
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Discussion |
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And my second question is, if it’s been used clinically in other areas, why hasn’t it been used clinically in cardiac surgery?
DR JAGGERS: Well, to answer your first question, I don’t know of any specific relationship, although in multiple models and multiple experiments in our laboratory, we have shown significant deterioration of pulmonary function and specifically dynamic compliance associated with bypass.
And it’s interesting that actually in a study that we presented several years ago, we used a total cardiopulmonary bypass model in which prevented any antegrade right ventricular flow to the lungs, and we actually showed a detriment in dynamic compliance and a decrease in endothelial dependent pulmonary vasorelaxation and an increase in pulmonary vascular resistance.
So in relationship to the previous authors, I think that in their model in which they had a very small amount of blood flow going to the lungs, that tends to be associated with worsened pulmonary function. We have seen that in our laboratory as well. It is unclear whether this is related to the ischemia or the reperfusion.
After constructing and carrying out this experiment, we subsequently found that a group, the lead author being Tisani, actually has given C1 inhibitor clinically in a randomized study in neonates undergoing the arterial switch procedure, and they were able to show that kallikrein and complement were effectively inhibited. They weren’t able to show a significant clinical difference except in the capillary leak syndrome and weight gain.
DR CHRISTOPHER A. CALDARONE (Toronto, Ontario, Canada): As a fellow participant in the large animal surgery research world, I can only applaud you for what must have been a very long and difficult study. One small question: why 19 controls?
DR JAGGERS: During the construct of this study, we actually designed this for fewer animals. In the process of doing this, we had a significant number of animals that were relatively unstable early on. And rather than excluding those animals, we elected to keep them within the group, and it just took more animals to do it. Interestingly enough, we had only 5 animals in the highest dose group, and we added that group, and the power analysis suggested that only 5 animals were necessary. So clearly we overpowered on the control side.
DR CALDARONE: Well, for translational research, that’s actually probably the better way to approach it. Inclusion of the animals that are unstable probably better reflects clinical reality. The other thing I wanted to ask is this. Tau is a measure of early diastolic function that usually reflects calcium handling events in early diastole, perhaps the first few milliseconds of diastole, whereas the end-diastolic pressure/volume relationship is usually more related to physical properties of the myocardium at the end of diastole where tissue edema and that sort of thing usually come into play. In light of the use of the C1 esterase inhibitor as a modulator of inflammation, it would seem logical to look at end-diastolic pressure/volume relationship to see whether there is an impact there.
DR JAGGERS: That’s a good point. We have previously shown in a study using soluble complement receptor inhibitor (SCR1), we showed that both tau and preload recruitable stroke work (PRSW) were favorably affected. In that study we looked at actually cellular calcium handling and the complement inhibition showed a benefit. I can’t explain why PRSW was not improved in this study and why tau is adversely affected in this model, because if one invokes the theory of increased myocardial edema from inflammatory injury one should see a deterioration of PRSW over time.
DR CALDARONE: That’s a very interesting finding.
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Acknowledgments |
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