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Eur J Cardiothorac Surg 2005;28:801-804
© 2005 Elsevier Science NL
Institute of Experimental Medicine, University of Cologne, Robert-Koch-Str. 10, Cologne 50931, Germany
Received 10 March 2005; received in revised form 26 August 2005; accepted 13 September 2005.
* Corresponding author. Tel.: +49 221 478 4129; fax: +49 221 478 6264. (Email: JH.Fischer{at}uni-koeln.de).
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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(10 µmol/l) was used for inducing contraction and substance P (10 nmol/l) for inducing EDR, which was calculated in percentage of the precontraction. Indomethacin (10 µmol/l) was added in all measurements to eliminate the influence of prostaglandins. In additional similar experiments (n
= 5), the influence of 1000 KIU/ml aprotinin on the EDR caused by the endothelium-derived hyperpolarizing factor (EDHF) was tested using L-NNA (300 µmol/l) to block all NO formation. Results: The EDR of pig coronaries (82 ± 5% or 80 ± 5% of the precontraction in the control tests before and after aprotinin exposure) was not significantly changed by 500 KIU/ml aprotinin (78 ± 7%). A small, but significant reduction of less than 1/10 of the EDR was induced by 1000 KIU/ml aprotinin (74 ± 5%). After accounting for L-NNA for NO blockage, no aprotinin-related difference remained (59 ± 6% vs 60 ± 6% in controls). Conclusion: For clinically relevant concentrations of aprotinin up to 500 KIU/ml, no significant reduction of the EDR can be found in epicardial coronary arteries of the pig. For higher doses of 1000 KIU/ml, a reduction in NO production seems to be the cause of the small but significant reduction of the EDR by aprotinin. Therefore, danger for impairment of coronary EDR by aprotinin at clinical dosage levels, as suggested by studies on rat aortas, seems to be absent in coronary arteries of a large mammalian model.
Key Words: Aprotinin • Endothelium-dependent relaxation (EDR) • Coronary artery • EDHF
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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On the other hand, fatal pulmonary thromboembolization during liver transplantation associated with aprotinin administration has been described [9]. Negative side effects could be found also for the kidney [10–12], especially at low body temperatures [13]. Moreover, an aprotinin-induced increase in the risk of graft thrombosis was found after myocardial revascularizations [14].
Ülker et al. [15] recently reported that aprotinin selectively impairs endothelium-dependent relaxation (EDR) as well as basal NO availability. Such an aprotinin-induced dysfunction would induce a high risk in cardiac operations and especially in coronary revascularizations, as the ability for endothelium-dependent relaxation (EDR) of the coronary system is of predominant importance for myocardial blood supply during and after cardiovascular interventions.
Are these results reproducible in other species and thus relevant for human coronary arteries? Ülker et al. [15] used vessel rings and cultured cells of rats. In their experiments only the cultured cells were of coronary origin, while the rings were from the thoracic aorta. Large reductions of EDR by aprotinin were reported, both for acetylcholine-induced EDR and for calcium-ionophore-induced EDR. But for all aprotinin concentrations from 125 to 500 KIU/ml, the magnitude of this effect was unchanged and had no correlation to the various concentrations.
Therefore, we decided to retest the findings of Ülker et al. [15] in a real coronary model of a larger non-rodent species. We used rings from the coronary arteries of the pig, a species that shows close similarities to the human, especially for the heart [16]. Instead of isometric vessel wall tension measurements, we used an isotonic lever transducer system to test the EDR-induced caliber changes of the vessels, which are really relevant for regulation of the coronary resistance.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Five-millimeter long rings were cut from the artery and tested for their contractile and EDR reactivity. These measurements of coronary function were carried out by means of an isotonic lever transducer system. The coronary rings were fixed between two triangular steel-wire holders under a load of 2 g, placed in a container with 10 ml Krebs–Henseleit solution (KH), and bubbled with 95% O2 + 5% CO2 (Carbogen) at 37 °C. This solution contains (in mmol/l): 143.1 Na+, 5.9 K+, 1.6 Ca2+, 1.2 Mg2+, 126.0 Cl–, 25.0 HCO3 –, 1.2 H2PO4 –, 1.2 SO4 2–, and 5.1 glucose.
The system records contractions or dilations of the coronary rings depending on the substance applied in the organ bath. Any alteration of vascular ring diameter under the constant distension load of 2 g was recorded (Multi-pen Recorder; Rikadenki Kogyo Co., Tokyo, Japan) via a transducer (Lever Transducer B 40 Type 373; Hugo Sachs Elektronik; March, Germany) and an amplifier (Transducer-Amplifier Module Type 705/1; Hugo Sachs Elektronik).
After reaching a steady state of the vascular diameter in a first run, KCl (60 mmol/l) was applied to trigger a maximal contraction (see Fig. 1
). All concentration values are the concentrations in the organ bath of 10 ml KH. Following each run, the KH was exchanged at least three times to remove all traces of the test substances. Each of the following runs was initiated by giving indomethacin (10 µmol/l) to block the cyclooxygenase pathway. The second run was then continued by adding PGF2 (10 µmol/l; Dinolytic, Pharmacia Upjohn GmbH, Erlangen, Germany), which induced contraction of the rings, followed by application of substance P (SP) (10 nmol/l, Fluka Chemie GmbH; Neu-Ulm, Germany), which caused endothelium-dependent dilation.
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In a separate group of rings (n = 5), all NO production was blocked by the addition of L-NNA (300 µmol/l N-nitro-L-arginine, Sigma-Aldrich GmbH, Steinheim, Germany), and the four run measurements were done including 1000 KIU/ml aprotinin in the third run. At the end of the test, Papaverin (200 nmol/l; Knoll, Ludwigshafen am Rhein, Germany) was added to achieve maximal dilation.
All data are expressed as mean values ± standard deviation (SD). Significance of differences between groups was tested using Student's t-test. Differences between several groups were tested using ANOVA followed by Bonferroni t-test. Statistical significance was considered as p < 0.05.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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and was changed non-significantly by 500 KIU/ml to 78 ± 7% (n
= 7). 1000 KIU/ml aprotinin induced a small but significant (p
< 0.05) reduction from 80 ± 5% to 74 ± 5% (n
= 6, see Fig. 2
). The second control after aprotinin exposure (82 ± 8% for the 500 KIU/ml group and 80 ± 5% for the 1000 KIU/ml group) was always within the range of the first control before aprotinin exposure. In experiments including L-NNA for NO blockage, the relaxation was reduced to 59 ± 6% in the controls and unchanged during incubation with 1000 KIU/ml aprotinin (60 ± 6%, n
= 5).
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without L-NNA continuously increased during the course of each experiment. The average values for all experiments (n
= 13) were 0.59 ± 0.29 mm for the first control. The contraction response significantly increased to 0.88 ± 0.26 mm during aprotinin exposure (p
< 0.05 in Bonferroni t-test) and further increased significantly to 1.04 ± 0.32 mm during control after aprotinin exposure (see Fig. 1). The average duration of measurements was 111 ± 42 min for the control measurements and 89 ± 17 min for the measurements during aprotinin exposure. |
4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Similar to our results, Allen et al. [17] have found that endothelium-dependent relaxations in human saphenous vein rings, explanted for coronary bypass, were unaffected after aprotinin treatment. The remaining question was whether the specific coronary arteries react in a different manner. Unfortunately, we were not able to perform our experiments on coronary rings from human hearts (for instance from explanted transplant recipient hearts), as this material is extremely inhomogenous and predamaged. Thus, we chose the coronary arteries of a large mammal whose heart resembles the human heart in many aspects from size to coronary distribution. We used undamaged material from the slaughterhouse to detect even slight reductions of EDR.
In our study, measurements with and without aprotinin were made on the same coronary ring with the control measurements made before and after aprotinin exposure. Thus, the risk of additional damage interfering with the results could be eliminated, especially by the second control measurement after aprotinin exposure. We used only one concentration of 10 nmol/l SP, which induces an 80% dilation in control experiments, the percentage of EDR for which Ülker et al. [15] reported the largest reductions to about 20–40% dilation induced by aprotinin. Thus, we cannot show the full concentration response curve, but we do clearly show that the 80% dilation induced by 10 nmol/l SP is uninfluenced up to 500 KIU/ml aprotinin and only marginally reduced by 1000 KIU/ml. We are therefore able to exclude substantial reductions of this dilation for the given EDR in our model.
These concentrations are higher than the current clinically used applications. As shown by Hardy and Desroches [18] in a clinical study, low dose applications of aprotinin (140 mg i.v. loading dose, 140 mg pump prime, and 35 mg/h i.v. constant infusion) result in plasma concentrations around 125 KIU/ml, while in high-dose protocols (double the doses given for the low-dose regime) about 250 KIU/ml is reached.
We used a standardized technique for precontraction of the rings with PGF2 and relaxation with substance P after incubation with indomethacin, in order to eliminate interfering prostaglandin effects. We preferred Substance P [19], which safely induces strictly endothelium-dependent relaxations by NO production [20] and by hyperpolarization (EDHF) [21] in any species, without a direct stimulation of the vascular muscles of large arteries [22]. Ülker et al. [15] used acetylcholine and calcium ionophore. But acetylcholine tends to increase vascular contraction with increasing concentrations in addition to EDR, and in its action it varies substantially in different species and organs [23,24]. Calcium ionophore A 23187 directly elevates endothelial Ca2+ in a receptor-independent fashion, bypassing any effect of receptor number or coupling [25].
Our technique of consecutive measurement of control, aprotinin exposure, and second control of the same ring yields safe results for the influence of aprotinin on the percentage of EDR versus first and second control. The percentage of EDR was unchanged between the first and the second control. But this technique also shows that the contraction of the vessel segments induced by the standardized concentration of PGF2 increased continuously during the measurements even though the EDR percentage (and thus also the endothelial reactivity) remained constant.
Ülker et al. [15] also found a change in the intensity of contractions of their vessel segments induced by phenylephrine, and they found differences in the contractile response with and without aprotinin. As they had no control measurement after aprotinin exposure, this lead to their speculation that aprotinin reduces the basal release of EDRF from the endothelial cells. But with respect to the lack of influence of L-NAME on this reduction in their experiments, this speculation seems to be rather unlikely.
In our experiments, the significant effect of the highest aprotinin concentration of 1000 KIU/ml was obviously induced by a reduced NO production as shown by the effect of high dose L-NNA. In this setup with blockage of NO production by L-NNA incubation, the differences between aprotinin and control measurements were eliminated leaving identical EDHF mediated dilations and proving that the differences without L-NNA had been NO-related. Thus, it seems to be clear that, despite the results from rats presented by Ülker et al. [15], no significant impairment of EDR by clinically relevant doses of aprotinin can be found in the coronary vessels of the pig. A risk for perioperative stenosis or formation of thrombosis provoked by an excessive inhibition of NO release—as concluded by those authors—could not be verified for the pig.
The question remains whether results from the rat aorta or the pig coronary artery are more relevant for the human coronary system. This can only be answered by a clinical study measuring the coronary flow with versus without aprotinin application. The feasibility of such a study will certainly be closely related to the further development of non-damaging CT or MRT flow measuring techniques in the future. In the meanwhile a damaging effect of aprotinin on the EDR of human coronaries seems to be less likely than claimed by Ülker et al. [15].
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Acknowledgments |
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Footnotes |
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Presented in part at the 32nd Annual Meeting of the German Society for Thoracic and Cardiovascular Surgery in Leipzig, February 23–26, 2003. Abstract published in Thorac Cardiovasc Surgery 2003;51:S1–98. |
References |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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