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Ann Thorac Surg 2003;75:210-215
© 2003 The Society of Thoracic Surgeons
a Department of Cardiothoracic and Vascular Surgery, Johannes Gutenberg-University, Mainz, Germany
b Department of Medicine-Cardiology, Johannes Gutenberg-University, Mainz, Germany
Accepted for publication June 21, 2002.
* Address reprint requests to Dr Buerke, Department of Medicine-Cardiology, Johannes Gutenberg-University, Langenbeck-Str. 1, D-55101 Mainz, Germany.
e-mail: buerke{at}mail.uni-mainz.de
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Abstract |
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METHODS: The effects of aprotinin on leukocyte–endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation and immunohistochemical analysis. The inflammatory cascade (leukocyte rolling, firm adherence, and transmigration) was studied after thrombin stimulation and after hemorrhage and reperfusion.
RESULTS: Intravenous bolus administration of aprotinin treatment (20,000 U/kg) significantly reduced leukocyte rolling from 55 ± 8 to 17 ± 3 cells/min (p < 0.01) and adherent cells from 12 ± 2 to 7 ± 1.4 cells (p < 0.05) along the venous endothelium of the rat mesentery after thrombin activation. In addition, aprotinin pretreatment significantly inhibited transmigration of leukocytes from 11.3 ± 1.2 to 6.0 ± 1.1 cells (p < 0.05) through the microvascular endothelial wall. Similarly, aprotinin decreased leukocyte–endothelium interaction after hemorrhagic shock. Moreover, immunohistochemistry demonstrated that aprotinin significantly attenuated P-selectin expression by the intestinal vascular endothelium.
CONCLUSIONS: Our data demonstrate that aprotinin potently inhibits recruitment of leukocytes in the microvasculature by interfering with endothelial cell–polymorphonuclear neutrophil interaction, and is a potent endothelial protective agent in clinically relevant doses. Thus, aprotinin pretreatment may be useful for primary prevention of inflammatory tissue injury mediated by ischemia–reperfusion injury such as shock, trauma, open heart operation, or other extensive vascular surgical procedures.
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Introduction |
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Until recently, little attention has been paid to the role of proteases in inflammatory tissue injury after ischemia and reperfusion. Elastase and cathepsin G are present as the two major neutral serine proteases in neutrophils. In this regard, elastase has the ability to degrade a variety of molecules such as elastin, type II and IV collagen, immunoglobulins, complement components, clotting factors, proteoglycans, fibronectin, and even intact cells [2]. Despite potent endogenous inhibitors such as 
1-antiproteinase inhibitor, 2-macroglobulin, or leukoproteinase inhibitor, elastase or cathepsin G may escape inactivation at sites of inflammation and still be able to cause tremendous tissue injury.
The serine protease inhibitor aprotinin belongs to the group of Kunitz protease inhibitors. Aprotinin has a molecular weight of 6,512 d and is a naturally occurring broad-spectrum serine proteinase inhibitor obtained from bovine lungs. Aprotinin is clinically used in cardiac operations owing to its efficacy in reducing postoperative blood loss [3] by blocking complement activation or fibrinolysis and inhibition of proteinases such as kallikrein, trypsin, and plasmin [4]. It has also been reported that aprotinin inhibits neutrophil elastase release [5] and superoxide anion formation [6], suggesting an inhibitory effect on neutrophil activation [7]. Other in vitro studies have demonstrated the important role of polymorphonuclear neutrophil elastase in reperfusion of ischemic bowel [8], in elastase-mediated damage of cultured vascular endothelial cells, and in isolated cardiac myocyte injury [9].
Neutrophil infiltration after reperfusion of ischemic tissue is known to be regulated by differentially expressed adhesion molecules (ie, selectins, integrins, and immunoglobulin superfamily members). P-selectin is involved in the early stages of the leukocyte–endothelial cell adhesion cascade, promoting leukocyte rolling, which enables adherence to the endothelium, subsequent transendothelial migration, and recruitment of leukocytes into injured tissue, which contributes to further tissue injury in inflammatory states [1, 10].
The aim of this study was to evaluate whether the serine protease inhibitor aprotinin could influence leukocyte–endothelial cell interactions and P-selectin expression in the rat mesenteric microvasculature after either acute inflammatory activation with thrombin or by physiologic activation with hemorrhage and reinfusion (ie, hemorrhagic shock).
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Material and methods |
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A loop of ileal mesentery was exteriorized through the midline incision and placed in a temperature-controlled fluid-filled Plexiglas chamber for observation of the mesenteric microcirculation by means of intravital microscopy [11]. The ileum and mesentery were superfused throughout the experiment with a buffered saline solution (containing in mmol/L: 140 Na+, 4.0 K+, 2.5 Ca2+, 1.0 Mg2+, 106 Cl-, 45 HCO3-) warmed to 37°C and bubbled with 95% N2 and 5% CO2. A microscope and a x40 water immersion lens (Zeiss, Goettingen, Germany) were used to visualize the mesenteric microcirculation and the mesenteric tissue. The image was projected by a charge-coupled device video camera onto a high-resolution monitor, and the images were recorded with a videocassette recorder. All images were then analyzed using computerized imaging software. Red blood cell velocity was determined on-line by using an optical Doppler velocimeter obtained from the Microcirculation Research Institute (College Station, TX). This method gives an average red blood cell velocity, which is digitally displayed on a meter, and allows for the calculation of shear rates. Red blood cell velocity (V) and venular diameter (D) were used to calculate venular shear rate (g) with the formula g = 8 (Vmean/D), where Vmean=V/1.6 [12].
The rats were allowed to stabilize for 20 to 30 minutes after the operation. After stabilization, a 30- to 50-µm-diameter postcapillary venule was chosen for observation. The number of rolling and adherent leukocytes was determined off-line by playback analysis of videotape taken from a video camera and videocassette recorder. Leukocytes were considered to be rolling if they were moving at a velocity significantly slower than the red blood cells. Leukocyte rolling is expressed as the number of cells moving past a designated point per minute (ie, leukocyte flux). A leukocyte was judged to be adherent if it remained stationary for more than 30 seconds [13]. Adherence is expressed as the number of leukocytes adhering to the endothelium per 100 µm of vessel length. To quantify the number of transmigrated leukocytes, the tissue area adjacent to the 100-µm length of postcapillary venule for a distance of 20 µm from the vessel wall was analyzed. The number of extravasated leukocytes was counted and normalized with respect to this area.
Thrombin superfusion protocol
Rats were randomly assigned to one of four groups: (1) control group (mesenteries superfused with buffer ± aprotinin intravenously), (2) rats treated with vehicle intravenously (1 mL NaCl 0.9%) before thrombin activation, (3) rats treated with 10,000 U/kg aprotinin intravenously before thrombin activation, and (4) rats injected with 20,000 U/kg aprotinin intravenously before thrombin activation. The amount of 20,000 U/kg aprotinin equals approximately 285 U/mL bolus dosage. In groups 2 through 4, the rats were subjected to superfusion of the mesentery with 0.5 U/mL thrombin. A baseline recording was made to establish basal values for leukocyte rolling, adherence, and transmigration (time 0). Immediately thereafter, thrombin superfusion of the mesentery was started. Video recordings were made initially and 30, 60, 90, and 120 minutes after initiation of superfusion for quantification of leukocyte rolling, adherence, and transmigration.
Hemorrhagic shock protocol
Rats were subjected to hemorrhage by withdrawal of blood to allow MABP to be maintained at 40 mm Hg for 60 minutes. Blood was collected in a heparinized syringe and kept at 37°C until reinfusion. Rats were then resuscitated by infusion of the shed blood. Rats were randomly assigned to one of the four experimental groups: (1) sham-operated rats, (2) hemorrhaged rats treated with vehicle (1 mL NaCl 0.9% 5 minutes before reperfusion), (3) hemorrhaged rats injected with aprotinin 10,000 U/kg 5 minutes before reperfusion, and (4) hemorrhaged rats injected aprotinin 20,000 U/kg 5 minutes before reperfusion.
Immunolocalization of P-selectin in the microvasculature
Immunohistochemical localization of P-selectin was determined in ileal samples after intravital microscopy was completed. A segment of ileum was isolated from the intestine and fixed in 4.5% paraformaldehyde in phosphate-buffered saline (pH 7.0). After dehydration, the sections were embedded in paraffin and cut into 5-µm tissue sections. Immunohistochemical localization of P-selectin was accomplished using the avidin–biotin immunoperoxidase technique (Vectasin ABC Reagent; Vector Laboratories, Burlingame, CA), using a monoclonal antibody against P-selectin (Pharmingen, Hamburg, Germany) exposed on the endothelial cell surface as described previously [14]. Fifty venules were analyzed per tissue section and five sections per group were examined per rat, and the percentage of positively staining venules was calculated.
Data analysis
All data are presented as mean ± standard error of the mean. Data were compared by analysis of variance using post hoc analysis with Fisher’s corrected protected least significant difference test. All data on leukocyte rolling, adherence, and transmigration, as well as on MABP and shear rates, were analyzed by analysis of variance, incorporating repeated measurements. Probabilities of 0.05 or less were considered statistically significant.
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Results |
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Superfusion of rat mesenteries with buffer alone for 120 minutes consistently resulted in a low number of rolling (12.5 ± 4 cells/min), adhering (1.2 ± 0.8 cells/100 µm vessel length), and transmigrated leukocytes (1.8 ± 0.8 cells/perivascular space [20 µm x 100 µm]). To exclude effects of aprotinin application, animals in sham experiments received aprotinin (10,000 or 20,000 U/kg). As shown in Figures 1 and 2, the higher concentration of aprotinin (ie, 20,000 U/kg) used in this study did not significantly change baseline values for leukocyte rolling, adherence, or transmigration.
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In rats superfused with buffer solution, only a small number of transmigrated leukocytes were observed at 120 minutes in the mesenteric extravascular space (within 20 µm from the postcapillary wall; Fig 2). However, in vehicle-treated rats superfused with 0.5 U/mL thrombin, the number of migrated leukocytes in the surrounding tissue was fivefold higher (p < 0.01). Pretreatment with 20,000 U/kg aprotinin before the study significantly attenuated the number of extravasated leukocytes after superfusion with thrombin. In contrast, the lower dose of aprotinin (ie, 10,000 U/kg) had only a partial effect.
Effect of aprotinin on hemodynamics after hemorrhagic shock
All groups of rats exhibited initial MABP values in the range of 110 to 125 mm Hg. In sham-operated control rats, MABP did not significantly change during the entire 150-minute observation period. Similarly, in hemorrhaged rats MABP was not significantly different. The heart rate was slightly elevated for 20 minutes after reperfusion, but the differences were not statistically relevant.
Venular shear rates for the four experimental groups of rats demonstrated no significant differences in initial shear rates among the four experimental groups of rats. After hemorrhage, shear rates in mesenteric venules abruptly decreased to less than 15% of the observed initial values. Therefore, the present hemorrhagic shock model is characterized by a marked hypoperfusion of the splanchnic microvasculature during the oligemic phase. However, on reinfusion of the shed blood, venular shear rates returned to nearly normal values.
Effect of aprotinin on leukocyte–endothelial interaction induced by hemorrhagic shock
A low baseline number of rolling (ie, 10 to 20 cells/min; Fig 3A) and adherent (ie, 1 to 3 cells/100 µm; Fig 3B)
were observed in the mesenteric microvasculature for all experimental group rats. However, the number of rolling and adherent leukocytes in untreated hemorrhaged rats exhibited a fourfold (p < 0.01) increase to 54 ± 6.2 cells/min and 6.3 ± 1.9 cells/100 µm, respectively, after reinfusion compared with normal control values (Fig 3). A similar pattern was observed in the case of leukocyte transmigration The number of migrated leukocytes in the surrounding tissue was significantly increased from 2.1 ± 0.8 to 9.1 ± 2.1 cells/perivascular space (20 µm x 100 µm; p < 0.01). In contrast, administration of aprotinin significantly inhibited the number of rolling and adherent leukocytes along the venular endothelium after blood reinfusion (Fig 3). These observed beneficial effects were dose-dependent, because 10,000 U/kg aprotinin resulted in only partial inhibition. A similar pattern was observed in the case of leukocyte transmigration (Fig 4).
Aprotinin treatment significantly attenuated the number of extravasated leukocytes from 9.1 ± 2.1 to 3.8 ± 1.9 cells/perivascular space (20 µm x 100 µm; p < 0.05). In addition, no significant change in the total number of circulating white blood cells was observed in the four experimental groups of rats, so that the changes in rolling and adherence could not be attributed to leukopenia after systemic aprotinin administration.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Leukocyte rolling, adherence, and subsequent transmigration through the endothelial wall of the mesenteric microcirculation are key steps in the inflammatory response known to occur in pathophysiologic states such as trauma, ischemia–reperfusion injury, and shock [15]. Leukocyte–endothelium interaction underlies a multistep process involving rolling, tight adhesion, and transmigration [16]. P-selectin, a member of the selectin family, mediates the rolling phenomenon of leukocytes along the vascular endothelial surface [8, 17]. Thus, rolling brings neutrophils in closer contact with the vascular endothelial wall, allowing firm adherence to occur. Subsequently, firm adhesion to the endothelium is mainly mediated by ß2-integrin interaction (ie, CD11/CD18) with intercellular adhesion molecule 1 [10]. After adhesion, activated polymorphonuclear neutrophils can undergo transendothelial migration [18].
Therefore, we raised the question of whether systemically administered aprotinin could directly affect expression of adhesion molecules. In this connection, we activated endothelial cells in vivo by either superfusing the rat mesentery with thrombin or by inducing whole-body ischemia–reperfusion. Acute endothelial dysfunction associated with enhanced leukocyte–endothelium interaction is a critical early pathophysiologic event mediated by P-selectin after thrombin stimulation and hemorrhage–reinfusion [19].
In this study, the degree of leukocyte rolling, adhesion, and transmigration after acute endothelial activation was determined in response to a rapid-onset stimulus (ie, thrombin superfusion) in vivo. Aprotinin significantly reduced both thrombin-mediated and hemorrhage-reinfusion–provoked P-selectin expression in mesenteric vasculature and therefore attenuated leukocyte rolling, adherence, and transmigration. Similarly, Asimakopoulos and colleagues [7] have demonstrated protective effects of aprotinin at the level of extravasation. However, we observed reduction of leukocyte rolling, adherence, and transmigration after aprotinin treatment, which might be explained by activation of the vascular endothelium with subsequent P-selectin expression, in contrast to direct leukocyte activation with N-formyl-Met-Leu-Phe [7]. We demonstrated that the inflammatory effects could be significantly inhibited dose dependently by intravenous aprotinin treatment. The beneficial effects in our study on leukocyte–endothelial interactions are probably mediated in part by diminished P-selectin expression on postcapillary venules in the thrombin-superfused mesentery, inasmuch as we could demonstrate significantly reduced levels of P-selectin expression on postcapillary venules in the aprotinin-treated groups.
Previous studies have demonstrated that complement activation can subsequently mediate P-selectin and intercellular adhesion molecule 1 expression [14]. In a previous study, we were able to demonstrate that aprotinin treatment was able to reduce myocardial reperfusion injury, which could be partly attributed to reduced complement activation [20]. Therefore, in the present study, aprotinin treatment might additionally be able to inhibit complement activation and subsequently P-selectin expression after hemorrhage and reperfusion.
Secondary to its direct endothelial protective effects, aprotinin may also inhibit the release of potent polymorphonuclear neutrophil chemoattractants such as elastase, cathepsin G, tumor necrosis factor-, and oxygen-derived free radicals [21]. In this manner, aprotinin has been shown to inhibit systemic tumor necrosis factor- release and CD11b upregulation [22]. Therefore, the administration of an exogenous protease inhibitor results in interruption of the protease-mediated inflammatory cascade.
In conclusion, these data support the concept that the serine protease inhibitor aprotinin effectively inhibits cell-surface expression of adhesion molecules (ie, P-selectin). This may by a key mechanism, besides the inhibition of complement activation, by which aprotinin could further inhibit leukocyte–endothelial interaction under inflammatory states. Aprotinin thus represents a potent pharmacologic agent useful for modulating the pathophysiology of leukocyte-induced endothelial dysfunction in inflammatory states, cardiopulmonary bypass, or perioperative management in cardiac operations.
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