Ann Thorac Surg 2008;86:568-575. doi:10.1016/j.athoracsur.2008.04.025
© 2008 The Society of Thoracic Surgeons
Original Articles: Adult Cardiac
Aprotinin Exerts Differential and Dose-Dependent Effects on Myocardial Contractility, Oxidative Stress, and Cytokine Release After Ischemia-Reperfusion
Matthew D. McEvoy, MDa,
Anna-Greta Taylor, MDa,
Juozas A. Zavadzkas, MDb,
Ira M. Mains, BSb,
Rachael L. Ford, BSb,
Robert E. Stroud, MSb,
Laura B. Jeffordsb,
Christy U. Beck, BS/BTb,
Scott T. Reeves, MDa,
Francis G. Spinale, MD, PhDa,b,c,*
a Department of Anesthesiology and Perioperative Medicine, Medical University of South Carolina, Charleston, South Carolina
b Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, South Carolina
c Ralph H. Johnson Department of Veterans Affairs Medical Center, Charleston, South Carolina
Accepted for publication April 9, 2008.
* Address correspondence to Dr Spinale, Division of Cardiothoracic Surgery, Medical University of South Carolina, 114 Doughty St, Rm 625, Charleston, SC 29403 (Email: wilburnm{at}musc.edu).
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Abstract
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Background: Cardiac surgery can result in left ventricular ischemia and reperfusion (I/R), the release of cytokines such as tumor necrosis factor, and oxidative stress with release of myeloperoxidase. Although aprotinin has been used in cardiac surgery, the likely multiple effects of this serine protease inhibitor limit clinical utility. This study tested the hypothesis that different aprotinin doses cause divergent effects on left ventricular contractility, cytokine release, and oxidative stress in the context of I/R.
Methods: Left ventricular I/R (30 minutes I, 60 minutes R) was induced in mice, and left ventricular contractility (maximal end-systolic elastance) determined. Mice were randomly allocated to 2 x 104 kallikrein inhibitory units (KIU)/kg aprotinin (n = 11), 4 x 104 KIU/kg aprotinin (n = 10), and vehicle (saline, n = 10). Based upon a fluorogenic assay, aprotinin doses of 2 and 4 x 104 KIU/kg resulted in plasma concentrations similar to those of the half and full Hammersmith doses, respectively.
Results: After I/R, maximal end-systolic elastance fell by more than 40% from baseline (p < 0.05), and this effect was attenuated by 2 x 104 KIU/kg but not 4 x 104 KIU/kg aprotinin. Tumor necrosis factor increased by more than 60% from control (p < 0.05) with I/R, but was reduced with 4 x 104 KIU/kg aprotinin. Myeloperoxidase increased with I/R, and was reduced to the greatest degree by 2 x 104 KIU/kg aprotinin.
Conclusions: Aprotinin influences left ventricular contractility, cytokine release, and oxidative stress, which are dose dependent. These results provide mechanistic evidence that multiple pathways are differentially affected by aprotinin in a context relevant to cardiac surgery.
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Introduction
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Cardiac surgery with cardiopulmonary bypass (CPB), often attendent with periods of myocardial ischemia-reperfusion (I/R), can result in a systemic and local inflammatory response, oxidative stress, and transient myocardial dysfunction. Aprotinin, a naturally occurring serine protease inhibitor, has been commonly used in this setting as it has been shown to favorably affect coagulation pathways and reduce blood loss and transfusion requirements [1, 2]. However, because aprotinin is a nonspecific serine protease inhibitor, multiple biological pathways and systems can be affected. The downstream consequences of aprotinin on these multiple systemic pathways has raised potential concerns about mechanisms of action and ultimately clinical outcomes [3]. For example, aprotinin has been shown to modify various markers of inflammation such as the release of cytokine tumor necrosis factor-
(TNF) and interleukin-6 (IL-6) [4, 5]. However, these effects are not uniformly observed and may be due to the aprotinin dose utilized [6]. For example, in a clinical study by Engleberger and associates [6], when aprotinin was added to the CPB priming solution, the degree of TNF receptor activation was unaffected in the early post-CPB period. Moreover, past studies have demonstrated that aprotinin may have myocardial protective effects in the context of I/R [6–10]. However, an integrative examination to determine whether and to what degree a mechanistic relationship exists between aprotinin administration on left ventricle (LV) contractile function, cytokine release, and oxidative stress with I/R has not been performed. Accordingly, this study was performed to test the central hypothesis that using clinically relevant aprotinin concentrations in an intact murine model of I/R, differential effects on LV contractility, cytokine release, and oxidative stress would occur.
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Material and Methods
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Overview
This study performed an integrative set of measurements in an intact murine model of LV I/R in which the aprotinin doses were computed to reflect clinical dosing protocols (Hammersmith) and administered before the induction of I/R. Plasma aprotinin concentrations were computed in this animal model to confirm that these aprotinin doses would reflect relevant plasma concentrations encountered clinically when utilizing the half or full Hammersmith aprotinin dosing protocol. Response variables from this protocol included in-vivo measurements of LV contractility, plasma levels of TNF and IL-6, and in-situ quantitation of oxidative stress by a myeloperoxidase (MPO) reaction.
Experimental Design
Instrumentation and protocol
Adult FVB mice (10 to 16 weeks old, 24 to 30 g weight) were induced, intubated, and maintained under isoflurane anesthesia (2%). The right carotid was exposed and a precalibrated four-electrode-pressure sensor catheter (1.4F, SPR-839; Millar Instruments, Houston, Texas) was placed in the LV. A left thoracotomy was then performed and a purse-string placed around the left anterior descending artery just distal to the bifurcation of the left main coronary artery using 6.0 polypropylene. The ligature was tightened to induce ischemia (30 minutes) and then released for reperfusion (60 minutes). At the end of the reperfusion period, the catheter was removed, blood was collected from the carotid, and decanted plasma stored for subsequent analysis. The heart was then perfused with ice-cold saline, the LV excised, and a circumferential section placed in cryogenic freezing medium for histochemical analysis. Six FVB mice served as reference controls for plasma and histologic measurements. All animals were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (National Research Council, Washington, DC, 1996).
For the in-vivo study, mice were randomly allocated to one of three treatment groups: vehicle (0.9% saline), 2 x 104 kallikrein inhibitory units (KIU)/kg aprotinin, or 4 x 104 KIU/kg aprotinin. Randomization was performed before instrumentation, and aprotinin or vehicle was administered immediately after the completion of baseline measurements. An additional 6 mice were anesthetized and instrumented, but did not undergo I/R, and were used for reference control purposes for the biochemical and histochemical studies. Using this targeted coronary occlusion model, we have demonstrated previously that the relative area at risk is 35% with a coefficient of variation of 5% [11, 12]. In a preliminary set of studies (n = 6), fluorescent microspheres (F-8838, molecular probes, 15-µm diameter, 7.5 x 104) of different emission spectra were injected at baseline, at 30 minutes of ischemia, and at 60 minutes of reperfusion by LV injection. Total LV regional myocardial blood flow fell to approximately 50% of baseline values with peak ischemia and returned to within baseline values with reperfusion. Thus, this murine model of coronary artery occlusion provided a transient period of low myocardial blood flow followed by a restoration of blood flow, and therefore allowed for the study of LV function in the context of I/R.
Aprotinin protocol and dosing validation
Aprotinin (10,000 KIU/mL) was used at doses of 2 x 104 KIU/kg and 4 x 104 KIU/kg to approximate plasma concentrations, which reflect aprotinin plasma concentrations encountered clinically when utilizing the half Hammersmith and full Hammersmith doses, respectively [9, 10]. It has been demonstrated previously that using aprotinin a 4 x 104 KIU/kg intravenous bolus reached plasma concentrations corresponding to the full Hammersmith clinical dosing protocol [9]. Furthermore, this aprotinin dosing schedule is similar to weight-based dosing regimens used in previous animal models to achieve representative plasma aprotinin activity [7]. Nevertheless, the present study administered aprotinin in a unique model of I/R, and therefore a procedure was developed to measure relative plasma levels of aprotinin.
For this approach, a fluorogenic substrate cleaved by the serine protease plasmin was utilized in an ex-vivo assay system. Specifically, the peptide sequence D-ala-leu-lys-7-amido-4-methylcomarin (A8171; Sigma, St. Louis, MO) at a fixed concentration of 10 nM, was mixed in a reaction buffer containing a 1:33 dilution of normal mouse plasma and incubated at 37°C for 15 minutes in the presence and absence of 7 ug/mL of plasmin (P1876 [3 U/mg]; Sigma). The fluorescence of this reaction was detected in continuous fashion (Fluostar Galaxy; BMG Labtech, Durham, NC) at an excitation/emission wavelength of 365/440 nm. The plasmin substrate, plasmin concentrations, and the incubation conditions were determined from preliminary dilution studies to yield peak performance as defined as that which yielded a consistent and stable fluorescence signal. This reaction solution was then incubated in the presence and absence of increasing concentrations of aprotinin (range, 0 to 560 KIU/mL) to generate a standardized enzyme activity-inhibition curve. The standard aprotinin inhibition curve, which was generated in triplicate, along with the 95% confidence interval for this standard curve is shown in Figure 1. This inhibition curve demonstrated the classical exponential decay, and was subjected to regression analysis, yielding a significant relationship between the reduction in fluorescence to aprotinin concentrations (r = 0.99, p < 0.001). The intra-assay coefficient of variation was 5% and the interassay coefficient was 9%. This aprotinin enzyme inhibition assay was utilized to extrapolate relative aprotinin plasma concentrations. Specifically, at the completion of the studies described in the subsequent paragraphs, plasma was prepared and incubated in the substrate/plasmin substrate, the relative fluorescence obtained, input into the standardized aprotinin inhibition curve, and an aprotinin concentration computed.
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Fig 1. Relative plasma concentrations of aprotinin were computed using a fluorogenic assay in which a specific peptide, cleaved by plasmin was utilized. Using fixed concentrations of the plasmin peptide and plasmin, a standardized curve could be generated with increasing concentrations of aprotinin. The specific reagents and incubation conditions are detailed in the text. An exponential aprotinin inhibition curve was generated and was then fit to an exponential regression equation. An excellent regression fit was obtained, as shown by the dashed lines (y = 4,071 ± 7,643 e–0.003x; r2 = 0.98, p < 0.0001), and the 95% confidence interval for the aprotinin assay is shown by the solid lines. The relative fluorescence and computed plasma aprotinin concentrations for the 2 x 104 KIU/kg dose (triangles) and the 4 x 104 KIU/kg dose (diamonds) have been superimposed on the standard curve (circles [error bars are the standard deviation from the estimate]).
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Left ventricular contractility measurements
After initial instrumentation and a 30-minute stabilization period, LV pressures and relative volumetric units were continuously recorded using a pressure-conductance unit (ARIA, MPCU-200; Millar Instruments, Houston, TX) and integrated electrical stimulation (DAQ, PV Analysis Software; Millar). The LV pressure and volume signals were integrated with an electrocardiographic signal and digitized (PowerLab; AD Instruments, New South Wales, Australia). The placement of the LV conductance catheter, validation procedures, and algorithm have been described previously [11, 12]. This included correction for parallel conductance and regular in vitro calibration. With continuous recording of the LV pressure-volumetric signal, the ventilator was suspended for several seconds and then restarted, which altered venous return and thereby LV preload. An adequate reduction in venous return was defined as an approximate, transient reduction in LV systolic pressure of 30%. This resulted in a family of LV pressure-volume loops in which definable points of the LV end-systolic pressure-volume relationship could be determined. Isochronal points from the recorded LV pressure-volume loops were used to compute the slope, which in turn was used to compute maximal LV elastance (Emax) [13]. In addition, the isochronal values for LV end-diastolic volume and stroke volume were obtained from the LV pressure-volume relationships and used to compute preload recruitable stroke work.
Plasma analysis
Plasma samples were assayed for TNF and IL-6 using an enzyme-linked multiplex suspension array (Luminex X500002Z15; Bio-Rad, Hercules, California). The relative fluorescence obtained for each cytokine was converted to an absolute concentration using standards that were included in each assay. The standards for TNF and IL-6 resulted in highly linear calibration curves (r2 = 0.9979, r2 = 0.9984, respectively; p < 0.05), and the sensitivity range for TNF was 0 to 400 pg/mL, and for IL-6 it was 0 to 150 pg/mL. The intra-assay coefficient of variation was approximately 15%. In addition, plasma was subjected to a mouse-specific, high-sensitivity cardiac troponin-I enzyme-linked immunoassay (2010-1-HSP; Life Diagnostics, Inc, West Chester, PA).
Myocardial myeloperoxidase histochemistry
To assess the degree of oxidative stress induced by the I/R protocol, relative content of MPO was assessed by immunohistochemistry. Left ventricle frozen sections (5 um) were briefly fixed in an ice-cold acetone solution for 5 minutes, and washed twice in phosphate-buffered saline. The sections were then incubated in a 1:100 dilution of a rabbit polyclonal anti-MPO (Abcam AB 535-500) for 1 hour, washed 3 times in phosphate-buffered saline, and then incubated with a 1:250 dilution of an anti-rabbit IgG (6101; Vector Labs, Burlingame, CA). Visualization of the primary antisera binding sites was performed using the 3',3'-diaminobenzidine-hydrogen peroxide substrate (Vector Labs). Negative controls were utilized in all staining protocols and included substitution with nonimmune antisera. The LV free wall was imaged (Axioskop-2; Zeiss, Thornwood, NY) and five transmural images at a magnification of x20 (Plan-Neofluar; Zeiss) were digitized (AxioCam MRc; Zeiss). The digitized LV images were quantified using a computer-assisted method, which has been optimized for histomorphometric measurements [11]. Briefly, after background subtraction, the digitized images were set to a 0% to 100% gray-scale reference, and regions corresponding to a threshold signal of 70% or greater were identified as positive MPO staining using a standardized algorithm (SigmaScan Pro 5; SPSS, Chicago, Illinois). The positive stained area was divided by LV myocardial sampling area (387 x 103 µm2/field sample), yielding a percent area of MPO staining. Reference control LV sections were included in all staining protocols and used to normalize the MPO values.
Data Analysis
All of the data collected in this study were coded and the code not broken until the conclusion of the study. The observers for the histologic measurements were blinded to the treatment assignments throughout. Left ventricle pressure and contractility were compared using a two-way analysis of variance (ANOVA) model. Single point measurements were compared between treatment and control groups using a one-way ANOVA. After the ANOVA, pair-wise comparisons were performed using unpaired t tests corrected for the number of comparisons. To examine the magnitude of change in LV contractility, with respect to I/R and aprotinin, the percent change from the respective baseline value was computed. The test statistic was set to an arithmetic value of 0, and comparisons performed using a t statistic. A similar approach was taken for TNF and MPO in which the comparative changes were computed from reference control values. All statistical analyses were performed using the STATA statistical software package (Stata Corp, College Station, Texas). Values of p less than 0.05 were considered to be statistically significant.
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Results
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A total of 40 mice were enrolled in the I/R protocol, with 9 mice dying before the final set of measurements. These mice died of arrhythmias during reperfusion and were equally distributed among the three treatment groups (vehicle, n = 3; 2 x 104 KIU/kg aprotinin, n = 3; 4 x 104 KIU/kg aprotinin, n = 4; 
2 analysis, p > 0.70). Thus, the final sample sizes were as follows: vehicle, n = 10; 2 mL/kg aprotinin, n = 11; and 4 mL/kg aprotinin, n = 10. Using the aprotinin plasma assay and standard curve shown in Figure 1, the computed aprotinin plasma concentration for the 2 x 104 KIU/kg aprotinin group was 180 ± 33 and for the 4 x 104 KIU/kg aprotinin group was significantly higher, 242 ± 25 KIU/mL (p < 0.05). Plasma troponin-I values were not detectable in non-I/R mouse plasma samples; and at the completion of the I/R protocol, they were 0.41 ± 0.04 ng/mL in the vehicle group, 0.55 ± 0.22 ng/mL in the 2 mL/kg group, and 0.38 ± 0.05 ng/mL in the 4 mL/kg group, with no differences amount groups (ANOVA, p = 0.77).
Baseline hemodynamic and LV contractility indices, examined using the statistical approach previously described, were equivalent among all three groups at baseline (before randomization) and therefore were pooled for data presentation and clarity. Baseline heart rate was 531 ± 6 beats per minute, which is consistent for this murine model under stable, ambient conditions [11, 12]. Heart rate was continuously monitored during the administration of vehicle or either aprotinin dose, and remained unchanged from baseline values. At the completion of the I/R protocol, heart rate was slightly lower from baseline in the vehicle group (494 ± 7 beats per minute, p < 0.05) and was similar to baseline in both the 2 and 4 x 104 KIU/kg aprotinin groups (513 ± 18 and 543 ± 9 beats per minute, respectively). Left ventricle peak systolic pressure fell from baseline (98 ± 2 mm Hg) at peak ischemia in the vehicle group and in the 4 x 104 KIU/kg group (85 ± 5 and 85 ± 4 mm Hg, respectively, p < 0.05), but not in the 2 x 104 KIU/kg group (91 ± 7 mm Hg). At 60 minutes of reperfusion, LV peak systolic pressure returned to within baseline values in all groups.
LV Contractility With I/R: Effects of Aprotinin
Left ventricular contractility as defined by Emax fell from baseline values (0.78 ± 0.06 mm Hg µL–1 · mg–1) by approximately 50% at peak ischemia in all groups (p < 0.05). Whereas Emax returned to within baseline values after 60 minutes of reperfusion in the aprotinin 2 x 104 KIU/kg group (0.59 ± 0.09 mm Hg µL–1 · mg–1), Emax remained significantly reduced in the vehicle and aprotinin 4 x 104 KIU/kg groups (0.39 ± 0.05 and 0.45 ± 0.06 mm Hg µL–1 · mg–1, respectively, p < 0.05). The relative changes in Emax at reperfusion as a function of baseline values is shown in Figure 2
and highlights the differences in this index of LV contractility between the vehicle and aprotinin groups. Preload recruitable stroke work, another index of LV contractility, followed similar trends with respect to I/R and aprotinin. Specifically, preload recruitable stroke work was significantly reduced from baseline values (0.85 ± 0.16 mm Hg/mg) after I/R in the vehicle and aprotinin 4 x 104 KIU/kg groups (0.39 ± 0.07 and 0.48 ± 0.11 mm Hg/mg, respectively, p < 0.05) but was similar to baseline in the aprotinin 2 x 104 KIU/kg group (0.78 ± 0.16 mm Hg/mg).
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Fig 2. (Top) The relative change in maximal left ventricle (LV) elastance (Emax), an index of LV contractility, was computed as a percent of baseline values after ischemia/reperfusion (I/R). A significant reduction in Emax occurred in the vehicle (n = 10) and 4 x 104 KIU/kg aprotinin group (n = 10). However, this depression in LV contractile function was ameliorated in the 2 x 104 KIU/kg aprotinin group (n = 11). (Middle) Plasma levels for the cytokine tumor necrosis factor-alpha (TNF-) were increased after I/R in both the vehicle and 2 x 104 KIU/kg aprotinin groups when computed as a percent change to reference control values. This I/R-induced TNF release was abrogated in the 4 x 104 KIU/kg aprotinin group. (Bottom) Myeloperoxidase (MPO) content, as assessed by immunohistochemistry, was increased significantly from control values in all I/R groups when compared with reference control values. However, MPO levels were reduced from vehicle values in the 2 x 104 KIU/kg aprotinin group. Absolute values for these indices are provided in the Results. (*p < 0.05 versus respective baseline/referent control; +p < 0.05 versus vehicle; #p < 0.05 versus 2 x 104 KIU/kg aprotinin). (Black bars = vehicle; striped bars = 2 x 104 KIU/kg aprotinin; diagonal striped bars = 4 x 104 KIU/kg aprotinin.)
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Cytokine Release With I/R: Effects of Aprotinin
Reference control plasma levels for the cytokine TNF were 24.5 ± 1.8 pg/mL and were not detectable for IL-6. After I/R, plasma TNF levels were increased by approximately twofold in the vehicle and 2 x 104 KIU/kg aprotinin groups (41.5 ± 4.6, 43.6 ± 4.3 pg/mL, respectively, p < 0.05), but remained within normal reference values in the 4 mL/kg group (24.4 ± 3 pg/mL). The relative changes in TNF levels as a function of reference control value are shown in Figure 2. With respect to IL-6, a strong signal was detected in the vehicle group (471 ± 56 pg/mL, p < 0.05 versus a referent control value of 0). The IL-6 values were similar to vehicle values in both the 2 and x 104 KIU/kg aprotinin groups after I/R (532 ± 201, 467 ± 96 pg/mL, respectively).
Myocardial Myeloperoxidase With I/R: Effects of Aprotinin
Representative histochemical staining for MPO is shown in Figure 3
for reference controls, after I/R in the vehicle and aprotinin groups. While minimal MPO staining could be detected in reference control LV sections, a significant increase in MPO staining occurred after I/R, and these relative changes are summarized in Figure 2. While MPO staining was increased in both aprotinin groups, the relative levels were reduced significantly in the 2 x 104 KIU/kg group. Upon inspection of higher power images, differences in the relative distribution of MPO staining could be appreciated between the vehicle and the 2 x 104 KIU/kg aprotinin group (Fig 3). Specifically, MPO staining appeared to be associated with interstitial cells in all I/R groups, most probably inflammatory cells and macrophages. However, MPO staining was diffusely distributed in and around these interstitial cells in the vehicle group whereas MPO staining was more punctuate and localized to intracellular compartments in the 2 x 104 KIU/kg group.
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Fig 3. Representative photomicrographs of left ventricle sections stained for myeloperoxidase (MPO) taken from (a) reference control; (b) after ischemia/reperfusion in the vehicle only group; (c) in the 2 x 104 KIU/kg aprotinin group; and (d) in the 4 x 104 KIU/kg aprotinin group. A significant and robust increase in MPO staining was observed in the vehicle group, and while evident in the aprotinin groups, appeared reduced. Quantitative results are presented in Figure 2. Higher power images of a left ventricle section taken from the vehicle group revealed (e) an egress of MPO staining from interstitial cells, whereas in the 2 x 104 KIU/kg aprotinin group (f), MPO staining appeared to be confined to the intracellular compartment of these interstitial cells. (Original magnification: a, b, c, d, x20; e and f, x63.)
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Comment
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Until recently, a major clinical indication for using aprotinin in cardiac surgery was for hemostatic purposes. Clinical and basic science reports have demonstrated that this serine protease inhibitor may have multiple effects in the context of myocardial I/R injury [4–7]. However, the majority of these past studies have either focused upon a single biological event such as cytokine release, or a functional response such as LV function. The present study addressed this issue through a set of integrative measurements in an intact murine model of I/R in which clinically relevant aprotinin doses were utilized. The new and unique findings of the present study were twofold. First, using an aprotinin dose that achieved plasma concentrations consistent with a half Hammersmith clinical dosing regimen (2 x 104 KIU/kg), LV contractility was improved and a determinant of oxidative stress reduced after I/R. Second, aprotinin utilized at an aprotinin dose that achieved plasma concentrations similar to a full Hammersmith clinical dose (4 x 104 KIU/kg) completely abrogated the release of the cytokine, TNF, but failed to provide protective effects on indices of LV contractile function or oxidative stress. These results further demonstrate the multiple effects of aprotinin in the context of I/R and that these effects are not uniform across dosing regimens.
Aprotinin and LV Function
Studies have examined the effects of aprotinin in intact animal systems with respect to LV systolic performance [7, 14, 15]. In these studies, utilizing a full Hammersmith aprotinin dose (4 x 104 KIU/kg), protective effects on regional and global systolic function have been demonstrated after brief periods of ischemia. However, these studies did not serially assess LV pressure-volume relationships to determine LV contractility, and different aprotinin doses were not incorporated into the study design. The present study demonstrated that despite a recovery in LV pressure development with I/R, intrinsic defects in myocardial contractility were present and could be ameliorated with a low aprotinin dose (2 x 104 KIU/kg), but not with a twofold higher dose of aprotinin. To more carefully examine the relevance of the aprotinin dosing paradigm used in the present study, actual plasma aprotinin calculations were also performed. That plasma aprotinin measurements have been directly assayed in an experimental model of I/R, rather than inferred, is a unique aspect of this study. In a clinical study, Beath and colleagues [16] utilized an ex-vivo approach to compute relative plasma aprotinin concentrations in patients undergoing cardiac surgery requiring cardiopulmonary bypass. In that study, the aprotinin plasma concentrations obtained in patients receiving either the full or half Hammersmith dose, at the time immediately after separation from cardiopulmonary bypass, were very similar to those obtained in the present study. However, it must be recognized that the volume of distribution, pharmacokinetics, and serine protease inhibitory profiles are likely to be different in the murine system than that of man. Nevertheless, the present study demonstrated that different plasma aprotinin concentrations, which are achieved in clinical applications, impart differential effects on LV contractility and cytokine release.
Aprotinin and Cytokines
It is well established that myocardial I/R is accompanied by the activation and subsequent release of a number of inflammatory mediators including TNF and the interleukins such as IL-6. While the effects of TNF and IL-6 are mediated through specific cognate receptors, the downstream consequences include the induction of a cascade of biological signaling molecules and the formation of mediators of oxidative stress. The exposure of the myocardium to TNF can result in a duality of responses with respect to contractile function and structure [17, 18]. Specifically, acute and robust exposure of the myocardium to TNF can elicit a positive effect on contractile function whereas prolonged exposure can result in diminished myocardial performance. Proteases, such as serine proteases, contribute to the amplification and processing of cytokines, and therefore aprotinin has been demonstrated to attenuate cytokine release in the context of I/R [4–6]. Tumor necrosis factor is produced in a membrane-bound form and requires proteolytic processing by specific proteases such as TNF convertase (TACE or ADAM-17) to yield a soluble TNF that can interact with the TNF receptor complex [17, 18]. Moreover, TACE in and of itself requires proteolytic processing to attain full catalytic activity [19]. In light of the fact that aprotinin is a nonselective serine protease inhibitor, it is likely that aprotinin modulates the signaling cascades that culminate in TNF expression and activation. It has been well established that aprotinin affects the activity of proteolytic pathways and systems in a concentration-dependent manner [20–22]. For example, at very low concentrations (< 100 KIU/mL) plasmin activity is inhibited, but at twice this concentration (> 200 KIU/mL), other proteases such as kallikrein and elastase can be inhibited. As these aprotinin concentrations can be easily reached and often exceeded using a full Hammersmith dosing protocol, then it is likely that a number of proteolytic pathways can be affected.
Aprotinin and Oxidative Stress
An important downstream consequence of I/R and the elaboration of mediators of inflammation is the formation of reactive oxygen species such as MPO. With I/R, an influx of inflammatory cells occurs, most notably neutrophils; this infiltration occurs early with I/R and is considered to be a cellular hallmark of myocardial injury and dysfunction [20, 10]. Closely integrated with this cellular infiltrate is the formation and release of MPO [22, 23]. In the present study, increased myocardial MPO occurred after I/R, consistent with the activation of inflammatory cells [20, 21]. The low dose of aprotinin (2 x 104 KIU/kg) reduced the relative content of MPO, suggesting a modification of inflammatory cell recruitment/activation with I/R. One potential mechanism for this effect is that this low dose of aprotinin appeared to prevent the release of MPO from preformed stores contained within interstitial inflammatory cells. The present study provides new evidence to suggest that the modification of MPO by aprotinin may not be entirely due to the suppression of cytokines.
Summary and Limitations
Aprotinin has been historically recognized for improving hemostasis during and after cardiac surgery requiring cardiopulmonary bypass and, until recently, formed an important pharmacologic tool in the postsurgical armamentarium. However, because aprotinin is a serine protease inhibitor, multiple biological pathways can be affected, some of which may be detrimental to specific physiologic processes [3]. The present study builds upon the current body of knowledge regarding aprotinin by demonstrating that myocardial specific effects such as contractile function and oxidative stress can be achieved without affecting more global processes such as systemic TNF release. These observations may hold clinical relevance with respect to more selective dosing strategies of aprotinin and potentially reducing undesired noncardiac effects. Specifically, the present study utilized aprotinin dosing strategies that achieved plasma concentrations consistent with those obtained in clinical studies using either the half or full Hammersmith protocols, and distinctly different responses were achieved. Using an aprotinin dose of 2 x 104 KIU/kg, which achieved plasma concentrations consistent with a half Hammersmith dose, local myocardial effects were achieved after a period of I/R. In contradistinction, a twofold higher dose of aprotinin (4 x 104 KIU/kg), which achieved plasma concentrations consistent with a full Hammersmith dose, reduced systemic cytokine release, but did not provide an improvement in LV contractility.
However, extending these findings to the clinical context must be done with caution and in light of inherent study limitations. First, this was an acute I/R study, and therefore the longer term effects of aprotinin on myocardial contractility, cytokine release, and oxidative stress remain to be determined. Second, a limited number of cytokines and markers of oxidative stress were evaluated, and how aprotinin may affect a larger portfolio of inflammatory markers and determinants of oxidative stress remains unknown. Third, while the present study measured relative aprotinin concentrations and utilized a clinically applicable dosing regimen, the measurements were performed in a murine model that did not simulate cardiopulmonary bypass per se, and therefore clinical extension of these plasma aprotinin concentrations must be done with caution. The mouse was utilized in the present investigation because we have developed a means to measure LV contractility in this model and because the mouse has been demonstrated to possess a cytokine cascade similar to humans [24–26]. Moreover, murine transgenic constructs exist in which the TNF pathway has been genetically altered [18, 27]. These TNF transgenic constructs could then be utilized in future studies to identify the mechanistic underpinnings for the effects of aprotinin in the context of I/R. These mechanistic studies regarding aprotinin are either not feasible or are problematic in a clinical context. The results of the present study underscore the importance of identifying the basic mechanisms of action and pathways affected by aprotinin to address the current clinical controversies and questions that surround the use of this pharmacologic approach [3, 24].
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Acknowledgments
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The authors acknowledge National Heart, Lung, and Blood Institute Grants PO1-HL-48788, RO1-HL-59165, and Research Service of the Department of Veterans Affairs (F. G. Spinale), and Research Fellowships from Bayer Pharmaceuticals and the Foundation for Anesthesia Education and Research (M. D. McEvoy).
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Abstract
Introduction
