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Second Department of Medicine, Johannes Gutenberg University, Mainz, Germany;
BASF Pharma, Ludwigshafen, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C1 esterase inhibitor is an endogenous inhibitor of the classical complement pathway which might be insufficiently active in situations like ischemia and reperfusion, since C1 esterase inhibitor can be inactivated by neutrophil released proteases such as elastase (12). Further, beside its inhibitory effect on the activated C1 complex (C1q, C1r, C1s), C1 esterase inhibitor has blocking potency on the mannose-binding lectin (MBL) pathway, the kallikrein system, the coagulation system, and the fibrinolytic system. Until now there was no specific inhibitor of the classical complement system available.
Therefore, the major purposes of this study were to determine the effects of a novel synthetic small molecule inhibitor of C1s on 1) complement activation, 2) myocardial tissue injury, and 3) neutrophil accumulation in a well-established rabbit model of myocardial ischemia and reperfusion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Determination of biochemical C1s and C1r activity.
Complement C1s and C1r proteases were purified from human and rabbit
plasma according to a procedure described by Lane et al.
(13). Enzyme activity was measured spectrophotometrically
using the artificial substrate Cbz-Gly-Arg-S-bzl (custom synthesis from
Polypeptide, Wolfenbuettel, Germany). Release of Bzl-SH was quantified
by adding an excess of 5,5'-dinitro-bis(2-nitrobenzoic acid) (no.
43760, Fluka, Buchs, Switzerland) and detection of yellow color
(
= 405 nm).
Determination of mannan binding protein-associated serine
protease 1 (MASP1) inhibition.
MASP1 protease was purified from human plasma by the method of Tan et
al. (14). Enzyme activity was detected
spectrophotometrically using the artificial substrate
Cbz-Gly-Arg-S-bzl. Bzl-SH release was quantified by adding an excess of
5,5'-dinitro-bis(2-nitrobenzoic acid) and by a colorimetric readout at
= 405 nm.
Determination of thrombin inhibition.
Thrombin was purified from human plasma (T8885, Sigma, Deisenhofen,
Germany). Potency of inhibitors was measured spectrophotometrically
using a standard chromogenic assay with the artificial substrate S2238
(Chromogenix, Molndal, Sweden) and spectrophotometric measurement of
yellow reaction product at = 405 nm.
IC50 curves of inhibitors C1s-INH-248
(peptidomimetic inhibitor, originally derived from thrombin inhibitor
D-Phe-Pro-Arg, BASF patent application EP00/027710, m.w.
520.5; Knoll, Ludwigshafen, Germany) and C1-INH in concentrations of
0.1 nM to 100 µM were determined routinely for C1s, C1r, MASP-1 and
thrombin after 10-min preincubation.
Complement-mediated SRBC lysis.
To determine the ability of C1s-INH-248 to block the classical complement pathway, we used an erythrocyte hemolytic assay as described previously (8). SRBC (Nobis, Endingen, Germany) were incubated with 0.1–20 µl rabbit serum. Absorbance in the presence of 20 µl rabbit serum was considered 100% of hemolytic activity. The complement activity of the other tubes was calculated by dividing the absorbance of each tube by the absorbance of 20 µl serum x 100 and expressed as the percent RBC hemolysis.
To compare the effect of the C1s-INH-248 or the C1 esterase inhibitor (C1-INH, Berinert, Aventis-Behring, Marburg, Germany) we incubated sensitized sheep erythrocytes with 10 µl rabbit serum in the presence of different concentrations of C1s-INH-248 (50 ng/ml to 0.1 mg/ml) or C1 INH (0.1–5 U/ml).
Experimental protocol and determination of myocardial necrosis
Adult male rabbits (2.3–3.6 kg) were anesthetized with sodium pentobarbital (30–60 mg/kg i.v.). An intratracheal cannula was inserted through a midline incision, and the rabbits were placed on intermittent positive pressure ventilation (TSE ventilator, TSE Bad Homburg, Germany). A polyethylene catheter was inserted into the right external jugular vein for additional pentobarbital infusion or for administration of C1s-INH-248, C1-INH, or its vehicle. A polyethylene catheter was inserted through the right femoral artery and positioned in the abdominal aorta for the measurement of mean arterial blood pressure. After a midsternal thoracotomy, the anterior pericardium was incised and a 3-0 silk suture was placed around the left anterior descending (LAD) coronary artery 5–8 mm from its origin. The electrocardiogram (ST segment elevation, heart rate) and mean arterial blood pressure were continuously monitored and recorded on a chart recorder every 20 min. The pressure rate index, an approximation of myocardial oxygen demand, was calculated as product of mean arterial blood pressure and heart rate divided by 1000.
Myocardial ischemia (MI) was induced by tightening the initially placed reversible ligature around the LAD so that the vessel was completely occluded. This was designated time zero. C1s-INH-248 (i.e., 0.1, 0.5, and 1 mg/kg body weight), C1-INH (100 and 200 U/kg body weight), or vehicle (saline) was given i.v. as a bolus 55 min after the coronary occlusion (i.e., 5 min before reperfusion (R)). Five minutes later (i.e., after a total of 60 min ischemia) the LAD ligature was untied, and the ischemic myocardium was reperfused for 3 h.
The rabbits were randomly divided into seven major groups. Following administration of C1s-INH-248 blood samples were drawn to determine C1s-INH-248 elimination with HPLC analysis following bolus administration. Sham MI+R rabbits were subjected to the same surgical procedures as MI+R rabbits, except that the LAD coronary artery was not occluded.
Determination of myocardial necrosis
At the end of the 180-min reperfusion period, the ligature around the LAD was again tightened. Fifteen milliliters of 0.5% Evans blue was rapidly injected into the left ventricle to stain the area of myocardium that was perfused by the patent coronary arteries. Immediately following this injection, the heart was rapidly excised and placed in cold saline. The right ventricle, great vessels, and fat tissue were carefully removed, and the left ventricle was sliced parallel to the atrioventricular groove in 3-mm-thick sections. The unstained portion of the myocardium (i.e., the total area at risk) was separated from the Evans blue-stained portion of the myocardium (i.e., the area not at risk). The area at risk was sectioned into small cubes (2 x 2 x 2 mm) and incubated in 0.1% nitro blue tetrazolium in phosphate buffer at pH 7.4 and 37°C for 10 min. The irreversibly injured or necrotic portion of the myocardium at risk that did not stain was separated from the stained portion of the myocardium (i.e., the ischemic, but nonnecrotic, area). The three portions of the myocardium (i.e., nonischemic, ischemic nonnecrotic, and ischemic necrotic tissues) were subsequently weighed and indexed. In additional animals myocardial tissue was taken to perform histologic analysis of tissue injury and neutrophil infiltration and immunohistochemical analysis for determination of C5b-9 deposition.
Plasma creatine kinase (CK) analysis
Arterial blood samples (2 ml) were drawn immediately before ligation and every 60 min thereafter. The blood was collected in polyethylene tubes containing 50 IU heparin sodium. Samples were centrifuged at 2000 x g at 4°C for 20 min, and the plasma was decanted for biochemical analysis. Plasma protein concentration was assayed using the biuret method of Gornall et al. (15). Plasma CK activity was measured using the method of Rosalki (16) and expressed as international units per gram protein.
Determination of myocardial myeloperoxidase (MPO) activity
The myocardial activity of MPO, an enzyme occurring virtually exclusively in neutrophils, was determined using the method of Bradley et al. (17) and modified by Mullane et al. (18) and described previously (6). One unit of MPO is defined as that quantity of enzyme hydrolyzing 1 mmol peroxide/min at 25°C.
Measurement of superoxide radical release from rabbit polymorphonuclear leukocytes (PMN)
The rate of superoxide anion production by PMN was measured spectrophotometrically by the reduction of ferricytochrome c (19). Isolation of rabbit PMN was performed using a Percoll density gradient. The final PMN pellet was resuspended in HBSS (Sigma). Five hundred-microliter samples of the rabbit PMN suspension containing 5 x 106 cells were preincubated with ferricytochrome c (100 µM; Sigma) in a total volume of 900 µl for 15 min at 37°C in 1.5 ml spectrophotometric cells in a spectrophotometer model. The PMNs were stimulated with 100 nM leukotriene B4 or PMA (100 nM) in a final reaction volume of 1.0 ml with either C1s-INH-248 (50 ng/ml to 0.1 mg/ml) or vehicle. The absorbance at 550 nm was measured every 30 s, and superoxide anion production (nanomoles per 5 x 106 PMNs) was calculated by dividing the absorbance of the samples by the extinction coefficient for the reduction of ferricytochrome c (21.1 mM-1 cm-1).
Analysis of PMN-endothelium interaction
Isolation of rabbit PMN was performed using 6% dextran for sedimentation and a Percoll density gradient. PMNs were collected from 62 to 80%. Isolated autologous PMNs were then labeled with a fluorescent dye (Sigma) according to the method of Yuan and Fleming (20).
Rabbit aortas were isolated and placed in warm, oxygenated Krebs Henseleit solution. Fat and connective tissue were removed, and the aortas were cut into rings 2–3 mm in length. The aortas were then opened and placed with the endothelial surface up into a cell culture dish filled with 3 ml Krebs Henseleit solution. To stimulate endothelial cells to increase their adhesiveness for neutrophils, aortic rings were activated with thrombin and subsequently coincubated with fluorescence-labeled neutrophils with or without C1s-INH-248. In addition, neutrophil were activated with leukotriene B4 to augment PMN adherence. Adhered PMNs were counted using epifluorescence microscopy (Zeiss, Gottingen, Germany) and expressed as PMNs per square millimeter.
Statistical analysis
All values in the text and figures are presented as the
mean ± SEM of n independent experiments. All data were subjected
to ANOVA followed by Fisher’s protected least significant difference
test. p 
0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory activity of C1s-INH-248 and C1-INH was analyzed on
a panel of related serine proteases (i.e., human C1r, C1s, MASP-1,
thrombin, and rabbits C1s). From the analysis of biochemical activity
it was evident that C1s-INH-248 had superior activity on human C1s
compared with C1-INH (i.e., 10 times more potent; Table I
). However, C1r was only affected by
both compounds in the micromolar range. In contrast to C1-INH,
C1s-INH-248 did not inhibit MASP-1. Neither inhibitor affected thrombin
activity. In addition, related serine proteases such as plasma
kallikrein, XIa, and XIIa were tested for inhibition by C1s-INH-248.
However, we did not observe inhibition to a significant extent
(IC50 = >10 µM; data not shown). C1s-INH-248
had superior activity on rabbit C1s compared with C1-INH (i.e., 7 times
more potent; Table I).
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Incubation of sensitized SRBC with rabbit serum resulted in a
concentration-dependent serum-induced hemolysis of the red cells (Fig. 1A). Ten microliters of rabbit
serum exerted 70–80% hemolytic activity. Coincubation of 10 µl
rabbit serum with C1s-INH-248 (50 ng/ml to 100 µg/ml) resulted in a
concentration-dependent inhibition of the hemolytic activity to almost
a complete inhibition at 5 µg/ml (Fig. 1B). These results
clearly demonstrate the efficacy of the C1s-INH-248 to inhibit
complement activation. Compared on equimolar basis C1s-INH-248
demonstrated superior potency (0.19 nM vs 1.6 µM for
IC50 of SRBC hemolysis).
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In the sham MI rabbits, we observed that an i.v. bolus administration of 1 mg/kg body weight C1s-INH-248 had no detectable effect on any of the measured hemodynamic, electrocardiographic, or biochemical variables. In the two groups of MI/R rabbits, there were no significant differences in any of the variables observed before coronary occlusion. After reperfusion, the ST segment decreased to nearly control values, since coronary reperfusion had been effective. In both MI/R groups were no significant differences of the pressure rate index readings, suggesting that C1s-INH-248 did not appear to alter myocardial oxygen demand (data not shown).
Effect of C1s-INH-248 on myocardial injury following reperfusion
To ascertain the effects of C1 inhibition on the degree of actual
myocardial salvage of ischemic tissue following reperfusion, we
measured the amount of necrotic cardiac tissue expressed as a
percentage of either the area at risk or the total left ventricular
mass. There was no significant difference in the wet weights of the
areas at risk between the two ischemic groups (Fig. 2). About 30% of the ischemic myocardium
became necrotic in the vehicle group when indexed to the area at risk
or 9% when indexed to the total left ventricle. However, in the
C1s-INH-248-treated ischemic-reperfused group, the amount of necrotic
tissue was <9 or 3%, respectively (p <
0.01). Therefore, C1s-INH-248 (1 mg/kg body weight) significantly
protected against necrotic injury in ischemic-reperfused rabbits
(Fig. 2).
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). After 3 h of reperfusion we
detected in the 0.1 mg/kg body weight C1s-INH-248-treated animals a
similar degree of necrosis compared with that in vehicle-treated
animals (33 vs 38% necrotic area/area at risk). Animals treated with
0.5 mg/kg body weight C1s-INH-248 showed a partial protective effect
(23% necrotic area/area at risk; Fig. 3, right).
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To confirm the preservation of ischemic tissue, we determined the
effect of C1s-INH-248 on CK activity, a biochemical marker of
myocardial tissue injury. In sham MI/R rabbits receiving C1s-INH-248,
the plasma CK activity increased slightly. In the two ischemic groups,
plasma CK activity increased slightly during the period of myocardial
ischemia. However, a marked washout of CK into the circulating blood
occurred in rabbits receiving vehicle. In contrast, MI/R rabbits
treated with C1s-INH-248 had significantly lower plasma CK activities
compared with vehicle-treated rabbits (p <
0.01; Fig. 4).
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Since accumulation of neutrophils in the ischemic region during
reperfusion has been thought to be one of the major mechanisms
responsible for reperfusion injury, we measured MPO activity as a
marker for neutrophil accumulation in the myocardium (Fig. 5). MPO activity was very low in
nonischemic myocardium of both MI groups (no significant difference).
However, MI rabbits receiving only the vehicle exhibited a slight
increase in MPO activity in the ischemic region. MPO activity in
the ischemic region of C1s-INH-248-treated animals tend to be lower
compared with that in vehicle-treated animals
(p < 0.05). MPO activity increased in the
necrotic portion to 1.31 ± 0.23 IU/100 mg tissue. In contrast,
C1s-INH-248-treated MI/R rabbits exhibited significantly lower MPO
activity in the necrotic myocardial tissue (0.40 ± 0.05 IU/100 mg
tissue; p < 0.001).
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Effects of C1s-INH-248 on number of circulating white blood cells
To determine whether C1s-INH-248 exerted any neutropenic effects
that could contribute to its cardioprotection, we counted circulating
white blood cells at the beginning and during the experimental period.
White blood cells counts did not change significantly over the course
of the experiment, and there were no
significant differences between these groups (Table III).
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To determine whether C1s-INH-248 exerted any inhibitory effects on PMN radical generation following activation with leukotriene B4 or PMA, we performed spectrophotometric analysis. The rate of superoxide anion production by PMN following leukotriene B4 or PMA activation did not change significantly when PMN were coincubated with C1s-INH-248 (50 ng/ml to 0.1 mg/ml). Similar results were observed in human PMN.
Effects of C1s-INH-248 on PMN-endothelium interaction
To determine whether C1s-INH-248 exerted any effect on PMN adhesion to the vascular endothelium, we performed an in vitro adhesion assay. Thrombin activation resulted in increased neutrophil adherence to the aortic vascular endothelium. However, PMN adherence was not affected by C1s-INH-248 (50 ng/ml to 0.1 mg/ml).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, cardioprotective effects of complement inhibition were demonstrated for the C1 esterase inhibitor (C1-INH), sCR1, and Abs directed against C5 or C5a (6, 10, 11, 21, 22). C1 esterase inhibitor blocks the classical complement pathway by binding to the activated C1 complex (C1q, C1s, C1r), which results in dissociation of the complex (23). Further, C1-INH can be inactivated by elastase released from activated neutrophils, human proteinase (3), plasmin, or thrombin (12, 24, 25). Thus, inactivation of C1-INH can occur locally in inflamed tissues such as ischemic reperfused myocardium. Therefore, administration of exogenous C1-INH was able to reduce myocardial reperfusion injury (6, 7, 8).
C1s-INH-248 is a small molecule (m.w. 520) nonpeptide inhibitor that is highly specific for C1s. Inhibition of C1s can occur for the activated and nonactivated complexes. C1s-INH-248 acts as reversible and competitive inhibitor of activated C1s. Since C1s-INH-248 is a really small molecule, it is able to inactivate C1 quickly, whereas C1-INH inhibitory activity can be augmented 10 times when preincubated for 60 min. In contrast to C1s-INH-248, C1-INH not only inhibits activated serine proteinases C1s and C1r, it is also a major inhibitor of activated factor FXII, as well as an inhibitor of kallikrein and activated factor XI (12, 23). Therefore, the previous observed cardioprotective effects cannot be solely attributed to complement inhibition. However, with the present study we were able to demonstrate with the highly specific C1s inhibitor the important role of the classical complement pathway following myocardial ischemia and reperfusion.
In this regard, we were able to demonstrate inhibition of complement-mediated red cell hemolysis by C1s-INH-248 and C1-INH following rabbit serum administration. However, C1s-INH-248 had on eqimolar basis a superior potency (IC50 = 0.19 nM vs 1.6 µM). From the in vitro testing it was evident that C1-INH blocks also the MBL pathway (i.e., MASP1, MASP2, lectin complement pathway). Administration of C1s-INH-248 did not inhibit MASP1, nor did it affect the MBL pathway (data not shown). Therefore, it is unlikely that activation of the MBL pathway plays a dominant role in our model.
However, accumulation of the first component of the classical complement pathway (i.e., C1q) has been demonstrated in the ischemic-reperfused myocardium and has been related to increased neutrophil accumulation in this area (8, 26). C1q binds to membrane particles, mitochondrial fragments, or other subcellular components of the ischemic tissue and is able to activate the complement cascade with subsequent generation of C3a, C5a, and C5b-9 (4). C5b-9 deposits were observed in myocardial tissue from patients with myocardial infarction (27) and in animal hearts following ischemia and reperfusion (10, 28). We observed in the present study, in accordance with other studies, significant deposition of C5b-9 following myocardial ischemia and reperfusion. However, C1s-INH-248 treatment retarded C5b-9 deposition and tissue injury. In a similar approach of myocardial ischemia and reperfusion in C6-deficient rabbits, reduction of myocardial necrosis was related to a reduction in C5b-9 deposition (29).
Our results clearly show that C1s-INH-248, when administered 5 min before reperfusion as bolus injection (0.1, 0.5, and 1 mg/kg body weight), markedly retards, dose-dependently, postreperfusion cardiac injury. The reduction in tissue injury exerted by C1s-INH-248 cannot be attributed to any hemodynamic effects, since the bolus injection did not alter the hemodynamic parameters. Similarly, C1s-INH-248 did not exert any anti-thrombin activity, since the bolus injection did not alter the activated partial thromboplastin time compared with vehicle (data not shown).
Treatment with C1s-INH-248 resulted, aside from its complement inhibitory effect, in reduction of neutrophil infiltration. Clearly, neutrophils are involved in myocardial ischemia-reperfusion damage in our model, since we observed significant increases in MPO activities in vehicle-treated ischemic myocardial tissue. The effects of C1s-INH-248, however, cannot be attributed to changes in circulating white blood cell counts, since bolus injection of C1s-INH-248 did not result in a significant drop of white blood cell counts. These data eliminate the possibility that C1s-INH-248 exerts cardioprotection due to neutropenia, a phenomenon known to reduce reperfusion injury (30). Further, C1s-INH-248 did not affect neutrophil radical generation following leukotriene B4 or PMA activation and did not directly inhibit PMN-endothelium interaction. Therefore, the direct anti-neutrophil potency of C1s-INH-248 cannot be attributed to the cardioprotective effects. Similar, in other experiments complement depletion with cobra venom factor resulted in significant inhibition of myocardial injury and reduced PMN infiltration (2).
Neutrophil adherence to the vascular endothelium is an early and
important event following reperfusion of ischemic myocardium that is
mediated by various adhesion molecules (31, 32). The
complement system stimulates neutrophil-endothelium interaction, since
C5b-9 and C5a induce rapid translocation of
P-selectin from Weibel-Palade bodies to the endothelial surface
(33, 34). In this regard, blocking of selectins with
either a mAb or a soluble sialyl
Lewisx-containing oligosaccharide reduced
myocardial reperfusion injury in cats (35, 36). Further,
C5a induces the synthesis and release of cytokines, including IL-1,
IL-6, and TNF-
, which can induce the expression of ICAM-1 or
E-selectin (37). In a previous study we could demonstrate
that blocking of complement activation results in reduced expression of
P-selectin and ICAM-1 on the vascular endothelium (8).
Therefore, it is most likely that the reduced neutrophil accumulation
following C1s-INH-248 treatment can be attributed to indirect reduction
of PMN-endothelium interaction.
In conclusion, we have demonstrated that in vivo administration of the small molecule C1 inhibitor C1s-INH-248 attenuates myocardial injury following ischemia and reperfusion. These protective effects could be in part attributed to reduced PMN accumulation as well as decreased C5b-9 deposition following C1s-INH-248 administration in the reperfused myocardium. Furthermore, these in vivo results demonstrate the important role of classical complement pathway activation for tissue injury in inflammatory states such as myocardial ischemia and reperfusion.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Buerke, Second Department of Medicine, Johannes Gutenberg University, Langenbeckstrasse 1, 55101 Mainz, Germany. E-mail address: buerke{at}mail.uni-mainz.de
3 Abbreviations used in this paper: C5b-9, MAC, membrane attack complex; C1s-INH-248, synthetic small molecule inhibitor of C1s; C1-INH, C1 esterase inhibitor; CK, creatine kinase; LAD, left anterior descending; MASP1, mannan binding protein-associated serine protease 1; MBL, mannose-binding lectin; MI, myocardial ischemia; MPO, myeloperoxidase; PMN, polymorphonuclear leukocytes; R, reperfusion; sCR1, soluble complement receptor 1; SRBC, sheep red blood cells.
Received for publication May 1, 2001. Accepted for publication August 31, 2001.
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-induced endothelial cell expression of E-selectin and ICAM- 1. J. Immunol. 155:1434.[Abstract]This article has been cited by other articles:
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  M. N. Busche, V. Pavlov, K. Takahashi, and G. L. Stahl Myocardial ischemia and reperfusion injury is dependent on both IgM and mannose-binding lectin Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1853 - H1859. [Abstract] [Full Text] [PDF]
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