Ann Thorac Surg 1997;63:74-77
© 1997 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Differing Effects of Aprotinin and 
-Aminocaproic Acid on Cytokine-Induced Inducible Nitric Oxide Synthase Expression
Gary E. Hill, MD,
Janice A. Taylor, BS,
Richard A. Robbins, MD
Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, and Department of Anesthesiology, University of Nebraska Medical Center; and Research Service, Omaha Veterans Affairs Medical Center, Omaha, Nebraska
Accepted for publication August 2, 1996.
 |
Abstract
|
|---|
Background. Cell expression of inducible nitric oxide synthase (iNOS) is increased by cytokines, resulting in high endogenous levels of nitric oxide. Expression of iNOS has been implicated in organ injury, including myocardial reperfusion injury. Serine protease inhibitors reduce cytokine-induced iNOS expression. The protease inhibitors aprotinin and 
-aminocaproic acid (EACA), used to reduce blood loss after cardiac operations, were evaluated in vitro on cytokine-induced iNOS expression and nitric oxide production.
Methods. A murine bronchial epithelial cell line was stimulated with a mixture of cytokines (tumor necrosis factor-
, interleukin-1ß, and interferon-
) with or without aprotinin or EACA. The resultant iNOS expression was asured by northern blot analysis, and nitric oxide production was assessed by cell supernatant nitrite levels.
Results. Nitrite concentrations in the supernatant were significantly increased after cytokine stimulation; they were not affected by any concentration of EACA but were significantly (p < 0.05) reduced by aprotinin. Aprotinin significantly (p < 0.05) reduced cytokine-induced iNOS expression, whereas EACA had no effect.
Conclusions. Aprotinin, but not EACA, reduces cytokine-induced nitric oxide production by inhibition of iNOS expression. Because increased endogenous nitric oxide levels secondary to iNOS activation have been implicated in organ injury, aprotinin may have clinical benefit compared with EACA when used for cardiac operations.
|
Introduction
|
|---|
Several therapies are available to reduce blood loss after cardiac operations requiring cardiopulmonary bypass (CPB). Drugs currently used for this purpose include aprotinin and -aminocaproic acid (EACA) [1]. Both aprotinin [1] and EACA [2] are serine protease inhibitors with demonstrated antiinflammatory properties. Aprotinin reduces neutrophil activation and cytokine (tumor necrosis factor-) release during and after CPB [3], as well as the adverse cardiovascular effects of an endotoxin infusion [4]. -Aminocaproic acid inhibits kinin and bradykinin (a known inflammatory mediator) release from high-molecular-weight kininogen by inhibition of the plasma protease kallikrein [2]. Kallikrein activation occurs during cardiac operations [5] and induces neutrophil activation and neutrophil elastase release [6]; these events are thought to be important in CPB-induced organ injury. These reports therefore suggest that, like aprotinin, EACA may demonstrate antiinflammatory activity [2].
Recent studies have demonstrated increased nitric oxide (NO) generation during CPB [7] and increased lung tissue expression of the cytokine-inducible form of NO synthase (inducible nitric oxide synthase, iNOS) immediately after CPB [8]. Moreover, NO-induced lung injury and bowel vascular injury correlate with tissue iNOS expression [9]; thus, NO organ cytotoxicity is generally believed to be secondary to elevated endogenous levels of NO derived from iNOS activation [9]. Activation of iNOS results in higher, nanomolar endogenous concentrations of NO, whereas constitutive NO synthase activation results in lower, picomolar endogenous NO concentrations [9]. In addition, elevated endogenous NO levels have been implicated in myocardial reperfusion injury [10]. Because lung injury and myocardial reperfusion injury may occur after CPB and because iNOS activation may contribute to these injuries [7–10], we compared aprotinin and EACA in vitro for the respective effects of these two protease inhibitors on cytokine-induced iNOS expression and subsequent NO production. Nitrite concentrations were measured in a murine bronchial epithelial cell culture line supernatant as an indication of NO production, as NO in an oxygen-containing aqueous solution is rapidly oxidized primarily to nitrite, NO2- [11].
|
Material and Methods
|
|---|
The murine lung epithelial cell line LA-4 was purchased from American Type Culture Collection (Rockville, MD) and cultured in six-well tissue culture plates (Costar, Cambridge, MA) with Ham's F-12 and 15% fetal calf serum until confluent. After washing three times, the LA-4 cells were cultured in serum-free Dulbecco's modified Eagle's medium. The cells were induced to express iNOS by stimulation with Cytomix, a combination of human tumor necrosis factor- (10 ng/mL), human interleukin-1ß (10 ng/mL), and murine interferon- (10 ng/mL) (Sigma, St. Louis, MO). Aprotinin (Trasylol; Bayer, Inc, West Haven, CT) or EACA (Abbott Laboratories, North Chicago, IL) was added 30 minutes before stimulation. Cell viability was assessed by trypan blue exclusion and was always greater than 95%.
|
Nitrite Determinations
|
|---|
Nitrite levels of the LA-4 cell culture supernatant were determined by converting nitrite to NO under acidic conditions, as described previously [12]. Nitric oxide was measured under a nitrogen stream using a chemiluminescence analyzer (Sievers model 270B; Sievers, Boulder, CO). The area under the curve was measured, and nitrite levels of the LA-4 supernatants were determined by comparison with a standardized curve constructed using solutions of known nitrite concentrations.
|
Northern Blot Analysis
|
|---|
The capacity of aprotinin or EACA to decrease cytokine-induced iNOS messenger RNA expression in the LA-4 cells was evaluated by extracting total cellular RNA from adherent cells using a modification of the methods of Chomczynski and Sacchi [13, 14]. Samples of RNA were applied to 1% denaturing agarose gels, electrophoresed, and blotted onto Hybond-N filters (Amersham, Arlington Heights, IL). The filters were hybridized with a phosphorus-32–labeled murine iNOS complementary DNA probe (1 x 106 cpm/mL) generated by random priming using a multiprime DNA labeling system (Amersham). The murine iNOS complementary DNA probe was the 1.8-kb complementary DNA fragment spanning the presumed coding region of the iNOS complementary DNA clone (NcoI fragment), generously donated by Drs Ziao-wen Xie and Carl Nathan [15]. The filters were washed at a final stringency of 0.1 x standard saline citrate at 55°C and were exposed to Kodak X-OMAT S film (Eastman Kodak Co, Rochester, NY) at -70°C for 1 to 3 days. After autoradiography, the filters were stripped and hybridized with a phosphorus-32–labeled glyceraldehyde 3-phosphate dehydrogenase complementary DNA. Autoradiographs were assessed by scanning laser densitometry. Specific iNOS messenger RNA levels were calculated as the ratio of iNOS to glyceraldehyde 3-phosphate dehydrogenase messenger RNA expression and were given as a percentage of the Cytomix positive control to compare iNOS messenger RNA levels between different experiments [13]. The "housekeeping gene" glyceraldehyde 3-phosphate dehydrogenase is consistently expressed, and its expression is not altered by cytokine exposure.
|
Statistics
|
|---|
All results are reported as mean ± standard error of the mean. Statistical comparisons were made by the two-tailed Student's t test using a paired test for paired data, and were confirmed by applying one-way analysis of variance. Significance was defined as p less than 0.05.
|
Results
|
|---|
Nitrite Levels
Nitrite concentrations in the culture supernatant of the LA-4 cells were significantly elevated (p < 0.05) in the presence of Cytomix. The addition of aprotinin (1,000 U/mL) reduced nitrite accumulation in the Cytomix-stimulated LA-4 cell culture supernatant (p < 0.05), whereas EACA (1 mg/mL) had no significant effect. Nitrite levels in preparations with aprotinin (1,000 U/mL) and media alone were less than 10 µmol/L (p < 0.05 compared with media cultured alone) (Fig 1
).
View larger version (26K):
[in this window]
[in a new window]
|
Fig 1. . Nitrite levels (mean ± standard error of the mean [SEM]) in LA-4 cell culture supernatant fluid after 24 hours. The aprotinin concentration was 1,000 U/mL; that of -aminocaproic acid was 1 mg/mL (n = 3, each condition). *p < 0.05 compared with media only.
|
|
Aprotinin at 500 and 1,000 U/mL produced a significant (p < 0.05) reduction of nitrite concentrations in the LA-4 cell culture supernatant, whereas lower aprotinin concentrations did not demonstrate any effect (Fig 2). -Aminocaproic acid had no significant effects at any concentration on LA-4 supernatant nitrite concentrations (Fig 3).
View larger version (32K):
[in this window]
[in a new window]
|
Fig 2. . Dose-response effect of aprotinin on Cytomix-induced increase in nitrite concentration in LA-4 cell culture supernatant after 24 hours (n = 3, each condition). *p < 0.05 compared with Cytomix-only cultured cells. (SEM = standard error of the mean.)
|
|
View larger version (22K):
[in this window]
[in a new window]
|
Fig 3. . Dose-response effect of -aminocaproic acid (EACA) on Cytomix-induced increase in nitrite concentration in LA-4 cell culture supernatant after 24 hours. No significant effect was found on supernatant nitrite levels at either concentration of EACA (n = 3, each condition). (SEM = standard error of the mean.)
|
|
|
Northern Blot Analysis
|
|---|
Consistent with the decrease in nitrite concentrations, iNOS messenger RNA expression was decreased in LA-4 cells cultured with Cytomix plus aprotinin (1,000 U/mL) (p < 0.05 compared with Cytomix-stimulated only). No effects were measured in cells cultured with Cytomix plus EACA (1 mg/mL) (Fig 4).
|
Comment
|
|---|
Inducible NO synthase induction results in much larger (nanomolar) levels of endogenous NO compared with the smaller (picomolar) levels resulting from the action of constitutive NO synthase [9]. Elevated endogenous NO levels cause airway edema, lung injury, and liver and myocardial reperfusion injury [9, 10]. Although cytokines strongly and synergistically activate iNOS, they inhibit constitutive NO synthase activity [16]. The cytokines tumor necrosis factor, interleukin-1, interleukin-6, and endotoxin are all released systemically during CPB in humans [17]. Consistent with systemic cytokine release, there is evidence of increasing systemic NO production during CPB [7] and increased lung iNOS expression immediately after CPB [8]. Because elevated iNOS expression is frequently reported in studies demonstrating the possibility that endogenous tissue NO levels are cytotoxic and cause organ injury and dysfunction [9], pharmacologic reduction in iNOS expression may be advantageous when used during an inflammatory state such as CPB [17].
These data demonstrate that aprotinin, but not EACA, inhibits cytokine-induced iNOS expression and subsequent NO production by murine bronchial epithelial cells. A mechanism for the inhibition of iNOS induction by aprotinin is suggested by recent studies [18, 19]. Inducible NO synthase transcription is regulated by the nuclear regulatory protein nuclear factor 
B (NFB) [19]. To exert its effects inside the nucleus, NFB in the cytoplasm must first be dissociated from its inhibitor, IB [18, 19]. Serine protease inhibitors inhibit the cytokine-induced degradation of IB, thus preventing NFB activation and subsequent iNOS induction [18, 19]. These data suggest that aprotinin, a protease inhibitor, inhibits NFB dissociation from its inhibitor, IB, thereby preventing NFB release and reducing iNOS transcription. This mechanism would be consistent with the data presented in this study. -Aminocaproic acid, although it is also a protease inhibitor, apparently does not possess this property.
Because blunting of the inflammatory response during CPB reduces the incidence of post-CPB myocardial ischemia and infarction [20], pharmacologic therapies that reduce CPB-induced inflammation are clearly advantageous [21]. Of the two drugs investigated in this study that are used to reduce post-CPB bleeding, only aprotinin demonstrates reduction of cytokine-induced iNOS expression with subsequent reduction of endogenous NO levels. The benefits of this action of aprotinin remain to be determined clinically, but may explain some of the antiinflammatory effects reported with aprotinin and other serine protease inhibitors [20].
|
Acknowledgments
|
|---|
Supported in part by a grant from the Bayer Corporation (G.E.H., R.A.R.) and a Department of Veterans Affairs Merit Review Grant (R.A.R.).
|
Footnotes
|
|---|
Address reprint requests to Dr Hill, Division of Pulmonary and Critical Care Medicine, Department of Anesthesiology and Internal Medicine, University of Nebraska Medical Center, 600 South 42nd St, Box 984455, Omaha, NE 68198-4455.
|
References
|
|---|
- Royston D. Blood-sparing drugs: aprotinin, tranexamic acid, and -aminocaproic acid. Int Anesthesiol Clin1995;33:155–79.[Medline]
- Kleniewski J, Blankenship DT, Cardin AD, Donaldson V. Mechanism of enhanced kinin release from high molecular weight kininogen by plasma kallikrein after its exposure to plasmin. J Lab Clin Med1992;120:129–39.[Medline]
- Hill GE, Alonso A, Spurzem JR, Stammers AH, Robbins RA. Aprotinin and methylprednisolone equally blunt cardiovascular bypass-induced inflammation in humans. J Thorac Cardiovasc Surg1995;110:1658–62.[Abstract/Free Full Text]
- Swartholm E, Haglund U, Ljungberg J, Hedner U. Influence of aprotinin, a protease inhibitor, on porcine E. coli shock. Acta Chir Scand1989;155:7 –13.
- Kongsgaard UE, Smith-Erichsen N, Geiran O, Amundsen E, Mollnes TE, Garred P. Different activation patterns in the plasma kallikrein-kinin and complement systems during coronary bypass surgery. Acta Anaesthesiol Scand1989;33:343–7.[Medline]
- Wachtfogel YT, Hack CE, Nuijens JH, et al. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation. Am J Physiol1995;268:H1352–7.[Abstract/Free Full Text]
- Ruvolo G, Greco E, Speziale G, Tritapepe L, Marino B. Nitric oxide formation during cardiopulmonary bypass. Ann Thorac Surg1994;57:1055–6.[Medline]
- Delgado R, Rojas A, Glaria LA, et al. Ca2+ independent nitric oxide synthase activity in human lung after cardiopulmonary bypass.Thorax1995;50:403–4.[Abstract/Free Full Text]
- Alican I, Kubes P. A critical role for nitric oxide in intestinal barrier function and dysfunction. Am J Physiol1996;270:G225–37.[Abstract/Free Full Text]
- Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young HH, Ignarro NJ. Role of 1-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am J Physiol1992;262:H616–20.[Abstract/Free Full Text]
- Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE, Byrns RE. Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from l-arginine. Proc Natl Acad Sci USA1993;90:8103–7.[Abstract/Free Full Text]
- Archer SA. Measurement of nitric oxide in biologic models. FASEB J1993;7:349–60.[Abstract]
- Robbins RA, Springall DR, Warren JB, et al. Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation. Biochem Biophys Res Commun1994;198:835–43.[Medline]
- Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem1987;162:156–9.[Medline]
- Xie QW, Cho HJ, Calaycay J, et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science1992;256:225–8.[Abstract/Free Full Text]
- Walter R, Schaffner A, Schoedon G. Differential regulation of constitutive and inducible nitric oxide production by inflammatory stimuli in murine endothelial cells. Biochem Biophys Res Commun1994;202:450–5.[Medline]
- Casey LC. Roles of cytokines in the pathogenesis of cardiopulmonary-induced multisystem organ failure. Ann Thorac Surg1993;56:S92–6.
- Kim H, Lee HS, Chang KT, Ko TH, Baek KJ, Kwon NS. Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-B. J Immunol1995;154:4741–8.[Abstract]
- Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-B/Rel in induction of nitric oxide synthase. J Biol Chem1994;269:4705–8.[Abstract/Free Full Text]
- Wendel HP, Heller W, Michel J, et al. Lower cardiac tropinin T levels in patients undergoing cardiopulmonary bypass and receiving high-dose aprotinin therapy indicate reduction of perioperative myocardial damage. J Thorac Cardiovasc Surg1995;109:1164–72.
- Hennein HA, Ebba H, Rodriguez JL, et al. Relationship of the proinflammatory cytokines to myocardial ischemia and dysfunction after uncomplicated coronary revascularization. J Thorac Cardiovasc Surg1994;108:626–35.[Abstract/Free Full Text]
This article has been cited by other articles:
|
|
|
|
 
D. T. Mangano, Y. Miao, A. Vuylsteke, I. C. Tudor, R. Juneja, D. Filipescu, A. Hoeft, M. L. Fontes, Z. Hillel, E. Ott, et al.
Mortality Associated With Aprotinin During 5 Years Following Coronary Artery Bypass Graft Surgery
JAMA,
February 7, 2007;
297(5):
471 - 479.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. T. Mangano, I. C. Tudor, C. Dietzel, and the Multicenter Study of Perioperative Ischemia Re
The Risk Associated with Aprotinin in Cardiac Surgery
N. Engl. J. Med.,
January 26, 2006;
354(4):
353 - 365.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Pearl, D. P. Nelson, S. M. Schwartz, and P. B. Manning
First-stage palliation for hypoplastic left heart syndrome in the twenty-first century
Ann. Thorac. Surg.,
January 1, 2002;
73(1):
331 - 339.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. C. Landis, G. Asimakopoulos, M. Poullis, D. O. Haskard, and K. M. Taylor
The antithrombotic and antiinflammatory mechanisms of action of aprotinin
Ann. Thorac. Surg.,
December 1, 2001;
72(6):
2169 - 2175.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. E. Hill
The Inflammatory Response to Cardiopulmonary Bypass-- Should It Be Treated?
Seminars in Cardiothoracic and Vascular Anesthesia,
September 1, 2001;
5(3):
229 - 235.
[Abstract]
[PDF]
|
|
|
|
|
|
|
S. Ulker, M. G. Cinar, U. Bayraktutan, and A. Evinc
Aprotinin impairs endothelium-dependent relaxation in rat aorta and inhibits nitric oxide release from rat coronary endothelial cells
Cardiovasc Res,
June 1, 2001;
50(3):
589 - 596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Royston
Hemostatic Drugs in Prothrombotic or Hypercoagulable States
Seminars in Cardiothoracic and Vascular Anesthesia,
November 1, 1997;
1(4):
376 - 394.
[Abstract]
[PDF]
|
|
|
Top
Abstract
Introduction
p < 0.05 compared with Cytomix-only cultured cells.