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Am J Physiol Heart Circ Physiol 287: H872-H877, 2004. First published March 18, 2004; doi:10.1152/ajpheart.01083.2003
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Thrombin inhibitor reduces myocardial infarction in apoE–/– x LDLR–/– mice

Anne-Louise Hemdahl,1,2 Erling Falk,3 Peter Thorén,2 and Göran K. Hansson1

1Center for Molecular Medicine, Department of Medicine, and 2Department of Physiology and Pharmacology, Karolinska Institute, SE-171 76, Stockholm, Sweden; and 3Institute of Experimental Clinical Research, Aarhus University Hospital, Skejby Sygehus, DK-8200, Aarhus, Denmark

Submitted 19 December 2003 ; accepted in final form 17 March 2004


   ABSTRACT
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METHODS
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We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE–/–) x LDL receptor-deficient (LDLR–/–) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE–/– x LDLR–/– mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 µmol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol·kg·–1min–1) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.

atherosclerosis; thrombosis; hypoxia; inflammation; apolipoprotein E; low-density lipoprotein receptor

ATHEROSCLEROSIS IS USUALLY the underlying cause of acute coronary syndromes including myocardial infarction (19, 20). Although extensive atherosclerotic plaques reduce blood flow by narrowing the lumen of the coronary artery, life-threatening complications of atherosclerosis are usually caused by occlusive thrombi on the plaque surface rather than obliteration of the lumen caused by the sheer size of the plaque itself (19, 20).

Analysis of coronary arteries in autopsy material from patients with myocardial infarction has revealed the presence of thrombi on culprit atherosclerotic lesions (9, 12). Sites of thrombosis exhibit damages of the plaque surface with plaque rupture (also called fissuring) in the majority of cases and endothelial erosion in a large minority of them (9, 12). Ruptured plaques exhibit signs of inflammatory/immune cell activation, tissue factor expression, matrix metalloproteinase secretion, collagenolysis, and reduction of the smooth muscle cells (19, 30). Although the process leading up to the fatal event remains incompletely understood, clinical and experimental findings imply vasoconstrictive and inflammatory molecules as precipitating factors (26). It has been proposed that transient ischemia caused by vasoactive peptides such as endothelin or angiotensin II may result in perturbed gene expression, overt cell injury, and thrombus formation, which could in turn lead to persistent coronary occlusion and infarction (5, 7, 17, 28). However, evidence is still lacking for several steps in this sequence of events leading from atherosclerosis to thrombosis and myocardial infarction. This is in part due to the lack of suitable animal models.

Gene-targeted mouse models have provided a wealth of information concerning the pathogenesis of atherosclerosis (6). To what extent such genetically hypercholesterolemic mice develop myocardial infarction remained controversial for several years. However, we have recently shown that mental as well as hypoxic stress can elicit myocardial infarction in atherosclerotic mice (7). Hypoxia caused immediate ischemia that could be prevented by an endothelin type-A (ETA) receptor antagonist; hence, it most likely involved endothelin-dependent vasoconstriction. The vasoconstrictive phase was reversible upon cessation of hypoxia, but an irreversible phase of ischemia developed later with signs of myocardial infarction including troponin T leakage (7). The present study was performed to characterize the response that follows hypoxic stress and leads to myocardial infarction. Our data indicate that the first reversible ischemic phase is followed by a second phase that involves thrombin generation and results in myocardial infarction.


   METHODS
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 METHODS
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Animals. Male apolipoprotein E-deficient (apoE–/–) x LDL receptor-deficient (LDLR–/–) mice (16, 34) on the C57/Bl6J background were obtained from M&B (Ry, Denmark) after they were weaned. The mice were fed a Western-type diet that contained cornstarch, glucose, sucrose, cocoa butter, cellulose, minerals, vitamin mix, 0.15% cholesterol, and 21% total fat (wt/wt) for 8 mo, which led to severe hypercholesterolemia with cholesterol levels of 27.8 ± 1.3 mmol/l (mean ± SE). The housing and care of the animals and all of the procedures used in this study were in accordance with national guidelines and were approved by the Stockholm North Ethical Committee on Animal Experiments.

Hypoxic stress. Mice were anesthetized by inhalation of 1.6 ± 0.2% isoflurane. The liquid isoflurane was infused with a precision pump into a small evaporator connected to a ventilatory mask. The concentration of isoflurane was continuously measured with a Datex Capnomac Ultima analyzer (Datex-Ohmeda; Helsinki, Finland). Anesthetized mice were exposed to hypoxia as previously described (7). In brief, the oxygen supply in the ventilatory mask was reduced stepwise (in 2-min increments) from 21 to 16, 14, 12, and finally 10%. After the hypoxic episode, the oxygen concentration was reestablished to 21% for 15 min before the mice were allowed to regain consciousness.

Drug treatment. At 10 min before hypoxic stress, when anesthesia reached steady state, one group (n = 21) was injected with a bolus dose (5.6 µmol/kg of body wt sc) of the thrombin inhibitor melagatran (mol wt 430; AstraZeneca; Mölndal, Sweden). For continuous drug administration, an Alzet pump (Durect 1003D) was implanted subcutaneously on the backs of the mice after induction of hypoxic stress; the pump released 20 nmol·kg–1·min–1 of melagatran. The bolus and infusion doses were based on pilot experiments to obtain plasma concentrations of ~2 µmol/l after 48 h. The dose used was two to three times higher than the therapeutic dose (0.1–0.7 µmol/l) that is used in patients receiving prophylaxis against venous thromboembolism (10, 32) but well below the dose associated with a range of side effects including inhibition of fibrinolysis, which has been observed at plasma levels of 3.8 µmol/l in rodents (14). In control apoE–/– x LDLR–/– mice (n = 23), PBS was used instead of melagatran. In the melagatran-treated group, 3 of 21 mice died, and in the PBS-treated group, 3 of 23 mice died after the hypoxic stress but before the termination of the experiment. These mice could not be included in the study.

ECG. A two-lead limb connection was attached subcutaneously to allow monitoring of the ECG during stress. Baseline recordings were started when the mice had reached anesthetic stabilization. After 10 min of hypoxic exposure, the ECG was recorded for another 15 min. ECG analysis was performed with PcLaB software version 5.0 (2).

Troponin T analysis. Peripheral blood was collected from the jugular vein at death. When the blood had clotted, serum was obtained by centrifugation at 5,500 rpm for 10 min. Serum troponin T was measured with an Elecsys 2010 Immunoassay analyzer (Roche Diagnostics). The troponin T analysis was considered positive if the concentration was ≥0.01 µg/l.

Immunohistochemistry and histology. At death, the right auricle was removed, and the heart was perfused from the left ventricle with 10 ml of PBS at a constant pressure of 100 mmHg. A 5-mm-thick cross-section of the heart stretching from the upper part of the ventricles to the middle of the ventricles was removed, fixed in 4% formaldehyde in PBS for 24 h, dehydrated, and paraffin embedded. Initially, preparation for immunohistochemical and histological staining was performed on 10 randomly selected samples from each group. However, it was necessary to exclude two samples from the melagatran-treated group and one from the PBS-treated group because they contained insufficient amounts of material for analysis.

In the specimens used for quantitative morphological analysis, two histological sections were collected and stained with elastin trichrome and two adjacent sections were collected for immunohistochemistry. The latter were deparaffinized and blocked with 10% normal goat serum (Vector) for 30 min. The sections were rinsed under tap water, and a rabbit anti-fibrinogen antibody (A0080, diluted 1:1,000; Dako; Roskilde, Denmark) was applied overnight at 4°C. Nonimmune rabbit IgG was used as a control. After samples were rinsed in PBS, a secondary goat anti-rabbit antibody (E0432, diluted 1:300; Dako) was applied for 30 min. The sections were washed with Tris-buffered saline and incubated with an ABComplex (K376, Dako), rinsed, exposed to an alkaline phosphatase substrate (SK-5100, Vector Labs; Burlingame, CA), and stained with Mayer's hematoxylin. The numbers of fibrinogen-positive cells were counted in blinded sections, normalized to the total section areas, and expressed as the number of positive cells per square millimeter. Cardiomyocytes were considered positive when the sarcoplasm of the cell was colored red. Infarction size was calculated from the number of fibrinogen-positive cells multiplied by the mean cardiomyocyte size as estimated from analysis of 30 cardiomyocytes in each of 17 hearts. This value was divided by the total heart cross-sectional area to obtain the relative infarction size (in %). The total section areas and cardiomyocyte areas were measured using a morphometry program as described elsewhere (24).

A detailed histological analysis was performed in an additional group of eight hypoxia-stressed apoE–/– x LDLR–/– mice in which the heart was sectioned from the aortic root to the apex. Four glass slides were collected with two 4-µm-thick sections on each. After a 50-µm interval, an additional set of 8 sections was cut. One slide from each cutting level was stained with elastin trichrome. As controls, the hearts of four apoE–/– x LDLR–/– mice not exposed to hypoxia were sectioned and stained in an identical way.

Statistical analysis. Values are presented as means ± SE. Differences between means were analyzed using the Mann-Whitney U-test and differences between proportions were examined by {chi}2-test. P < 0.05 was considered statistically significant.


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Hypercholesterolemic apoE–/– x LDLR–/– mice were exposed to acute hypoxia at the age of 8 mo. At this stage, all mice had significant coronary atherosclerosis, but no increased mortality compared with wild-type mice was observed. A subgroup of apoE–/– x LDLR–/– mice was pretreated with the thrombin inhibitor melagatran before being exposed to hypoxia, and other animals were injected with PBS. All mice were monitored by ECG during the hypoxic stress and were analyzed for serum levels of troponin T when killed 48 h after the hypoxic challenge. Groups of mice were analyzed by histology and immunohistochemistry to assess the extent of myocardial infarction. A separate group of hearts from hypoxia- and non-hypoxia-stressed apoE–/– x LDLR–/– mice was serially sectioned and assessed for arterial thrombus formation.

ECG. Upon induction of hypoxia in the atherosclerotic mice, myocardial ischemia was manifested by typical elevations in the STU segment similar to those observed in humans suffering from myocardial ischemia (Fig. 1A). No difference in the immediate response to hypoxia was seen between the melagatran- and PBS-treated groups (Fig. 1B). Both groups displayed STU deviations starting at 16% oxygen concentration. In total, the duration of gradual hypoxia lasted for 10 min. The ECG changes declined immediately after cessation of hypoxia, and the STU alterations were back to normal after 10 min of normoxia, which demonstrates a reversible ischemic stress rather than a sustained myocardial infarction. Melagatran-treated apoE–/– x LDLR–/– mice responded similarly to hypoxic challenge as PBS-treated apoE–/– x LDLR–/– mice, which indicates that the acute STU changes were not a result of thrombin generation.



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  		Fig. 1. ECG changes in atherosclerotic mice during acute hypoxic stress. A: registrations from bipolar subcutaneous limb leads during stepwise induction of hypoxia accomplished by reducing the oxygen content in the ventilator supplying the anesthetized mouse. A prominent STU elevation was registered at 10% oxygen concentration and disappeared upon return to normoxia. B: acute hypoxia-induced elevations in the STU segment of the ECG. This elevation was of similar magnitude in melagatran- and PBS-treated control mice and declined when the oxygen level was normalized to 21%. Curve shows the change from baseline (values are means ± SE). ApoE/LDLR ko, apolipoprotein E-deficient x LDL receptor-deficient (knockout) mice.

 
Infarction marker analysis. Troponin T is a well-established serum-assay marker for detecting leakage from irreversibly damaged myocytes as a diagnostic test for myocardial infarction (25). At 48 h after hypoxia, mice of the melagatran-treated group displayed significantly lower troponin T levels than those of the PBS-treated group (P = 0.04; Fig. 2). Furthermore, in mice with troponin T levels above baseline values, the mean concentration was higher in the PBS-treated group (118.7 ± 43.9 ng/l in 10 of 20 mice) than in the melagatran-treated group (35.0 ± 25.0 ng/l in 4 of 18 mice; P < 0.05), which suggests that the average infarction size was smaller in the latter group (21).



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  		Fig. 2. Reduced troponin T levels in melagatran-treated mice. Serum concentrations of troponin T were measured 48 h after hypoxic stress to assess myocardial infarction; they were significantly lower in apolipoprotein E-deficient x LDL receptor-deficient (apoE –/– x LDLR –/–) mice that received melagatran before hypoxic stress (n = 18; 7.8 ± 6.1 ng/l) compared with the PBS-treated group (n = 20; 59.4 ± 25.3 ng/l; P < 0.05).

 
Histological analysis of infarction. Serial sections of hearts from hypoxia-stressed mice were analyzed for infarctions by immunohistochemistry and histology. Accumulation of the plasma protein fibrinogen in cardiomyocytes was analyzed as a hallmark for early irreversible cell membrane damage (Fig. 3; Refs. 13, 15, 29). Fibrinogen was previously detected within necrotic myocardial fibers as early as 3 h after coronary occlusion (13). Therefore, fibrinogen staining identifies infarctions also in early stages and well before leukocyte accumulation and cardiomyocyte necrosis can be observed. Mice that received melagatran before hypoxic stress displayed a significantly lower number of fibrinogen-positive cells than PBS-treated mice (80 ± 15 vs. 153 ± 34 cells/mm2; P < 0.05; Fig. 3). Furthermore, infarct size (infarction area divided by total heart area) was significantly reduced (by 46%) in melagatran-treated compared with PBS-treated mice (2.5 ± 0.5 vs. 5.4 ± 1.1%; P < 0.05; Fig. 3). Elastin-trichrome staining revealed myocardial scar formation (focal fibrosis) indicative of old infarctions in 8 of the 17 analyzed hearts. Old infarctions were manifested by scarred myocardial tissue (Fig. 4A) and recent infarctions (~48 h; Ref. 22) were distinguished by degraded myocytes with infiltration of inflammatory cells in the extracellular matrix (Fig. 4B). In contrast with the fibrinogen-stained recent infarctions, the incidence of old infarctions did not differ between melagatran-treated and control mice (n = 3 of 8 and 5 of 9 mice, respectively; P = not significant). Infarctions in apoE–/– x LDLR–/– mouse hearts were irregularly shaped and were located subendocardially or intramurally (Fig. 4B). No transmural infarctions were detected.



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  		Fig. 3. Immunohistochemical detection of myocardial infarction in mice. A: positive fibrinogen staining (red) of a recent myocardial infarction in the left ventricular wall of a PBS-treated mouse. B: adjacent control section where the primary antibody against fibrinogen has been replaced by nonimmune IgG. Both micrographs are shown at original magnification, x400. C: quantitative analysis of infarction size. Fibrinogen-positive cells were counted in a cross-section of the entire heart of each mouse. There were significantly fewer in the melagatran-treated group (n = 8; 80 ± 15 cells/mm2) compared with the control group (n = 9; 153 ± 34 cells/mm2; *P < 0.05). D: infarction area compared with total heart area was reduced in mice treated with melagatran (n = 8; 2.5 ± 0.5%) compared with control mice (n = 9; 5.4 ± 1.1%; *P < 0.05).

 


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  		Fig. 4. Histology of the heart in hypoxia-stressed apoE–/– x LDLR–/– mice. Paraffin sections were stained with elastin trichrome, which rendered elastin black, collagen blue, and muscle and blood components red. A: scarred myocardial tissue (focal fibrosis); original magnification, x200. B: recent myocardial infarction with degraded myocytes and infiltrating inflammatory cells. Infarcted area is indicated by dotted line; magnification, x200. C: advanced atherosclerotic plaque in an intramural artery; magnification, x400. D: an atherosclerotic plaque (thick arrow) with a superimposed thrombus (thin arrow) in an intramural artery; magnification, x600. E: intramural artery with atherosclerotic plaque displaying intraplaque hemorrhage; magnification, x200. F: erythrocytes (thick arrows) and foam cells (thin arrow) are found intermixed in the hemorrhaged plaque; magnification, x400.

 
Atherosclerosis and thrombosis. In a separate group of eight apoE–/– x LDLR–/– mice exposed to hypoxic stress, hearts were embedded in paraffin and serially sectioned for analysis of atherosclerosis and thrombosis. All of these hearts showed extensive atherosclerosis in the proximal coronary arteries (Fig. 5), and four of them displayed extensive atherosclerotic plaques even in the intramural arteries (see Fig. 4C). In two arteries, we detected severe atherosclerosis with superimposed thrombosis (Fig. 4D). In a coronary artery with obstructive atherosclerosis, intraplaque hemorrhage was evident with erythrocytes incorporated in the plaque and intermixed with foam cells (Fig. 4, E and F). Examination of serial sections from the hearts of control apoE–/– x LDLR–/– mice not exposed to hypoxic stress revealed atherosclerotic lesions in their coronary arteries, but there were no thromboses (data not shown).



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  		Fig. 5. Advanced atherosclerosis in apoE –/– x LDLR –/– mouse. Micrograph shows the aortic root and proximal left coronary artery (LCA) with advanced plaques. Elastin-trichrome staining; magnification, x50.

 

   DISCUSSION
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The present report shows that thrombin inhibition reduces myocardial infarction in atherosclerotic mice. Administration of the thrombin inhibitor melagatran reduced serum levels of the infarction marker troponin T as well as the number of fibrinogen-positive cardiomyocytes in mice exposed to hypoxic stress. These findings imply that thrombin is involved in the process leading from coronary atherosclerosis to myocardial infarction in this model and are compatible with the notion that thrombosis in atherosclerotic coronary arteries causes irreversible ischemia and infarction and provide an opportunity to study these responses in a murine model.

Our previous studies have shown that hypoxic as well as mental stress can elicit myocardial infarction in atherosclerotic mice (7). Hypoxia may trigger the procoagulant pathway and induce expression of tissue factor and plasminogen activator inhibitor-1 (4, 18, 23). This is likely to be particularly important in the atherosclerotic coronary artery, which has reduced fibrinolytic activity and increased propensity for thrombosis (1, 27, 33).

Thrombin is a key enzyme of the coagulation pathway by cleaving fibrinogen to fibrin (3). In addition, thrombin promotes adhesion and aggregation of platelets and adhesion and recruitment of monocytes and T cells (8, 11, 31). It also elicits endothelial activation including shape and permeability changes, mobilization of adhesive molecules to the endothelial surface, and stimulation of platelet-activating factor and cytokine production (3, 8, 11, 31). However, it remains to be determined whether the major target of thrombin in the present infarction model is to activate humoral coagulation, platelet activation, endothelial activation, or a combination thereof.

Hypoxic stress elicits transient endothelin-dependent myocardial ischemia in atherosclerotic mice (7). Ischemia-induced ECG changes were not affected by melagatran, which implies that thrombin is not involved in the immediate response to hypoxia. Instead, this early phase is likely to depend on endothelin-induced vasoconstriction (7). However, thrombin inhibition reduced infarction incidence at 48 h after hypoxia by 60% and significantly reduced troponin T release and infarction size. This suggests that hypoxic stress causes myocardial infarction through a two-phase pathway that involves an acute hypoxia-induced, endothelin-dependent phase that is transient and a subsequent thrombin-dependent, irreversible ischemic phase that leads to myocardial infarction.

Coronary atherothrombi were observed in some of the infarcted hearts. This is compatible with the notion that plaque activation leads to rupture, which can serve as a site for thrombus formation (9, 12, 30). Unfortunately, no quantitative analysis of activated plaques could be performed in the present study because only a few coronary atherothrombi were detected at death 48 h after hypoxic stress. The low incidence of coronary thrombi may be at least partly due to the high fibrinolytic activity in mice. However, one could speculate that the initial endothelin-dependent vasoconstriction leads to activation and/or damage of the endothelium that covers atherosclerotic lesions. Such sites might be preferential targets for thrombus formation, which would ultimately obstruct blood flow through the affected coronary artery.

Murine models have become indispensable in research on the pathogenesis of human diseases. However, they are limited by differences in organ size, vascularization, metabolism, and other factors between species. It should also be emphasized that the genetically targeted hypercholesterolemic mouse reaches plasma cholesterol levels that are beyond those seen in human hypercholesterolemic patients. The atherosclerotic process is also excessive in these mice: the time from disease initiation to infarction is months rather than decades (as is usually the case in humans). Finally, hypoxic ventilation under general anesthesia does not mimic any situation in which humans develop myocardial infarction. For all of these reasons, data from models such as the present one should be interpreted with caution, and findings cannot be applied directly to human disease.

In conclusion, the present study shows that hypercholesterolemic atherosclerotic mice develop myocardial infarction through a thrombin-dependent pathway in response to hypoxic stress. This suggests that thrombus formation mediates the development of myocardial infarction in atherosclerotic coronary arteries. The present model should be useful for investigators studying the pathway leading from coronary atherosclerosis to myocardial infarction.


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This work was supported by grants from AstraZeneca, the Swedish Heart-Lung Foundation, the Swedish Medical Research Council (6816 and 4764), and Wallenberg Consortium North.


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P. Thorén has been employed by AstraZeneca since 2001 and holds stock options.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Stefan Carlsson (AstraZeneca, Mölndal, Sweden) for kindly providing melagatran and Birgitte Sahl (Aarhus University Hospital, Denmark) for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. K. Hansson, Dept. of Medicine, Center for Molecular Medicine L8:03, Karolinska Hospital, SE-171 76, Stockholm, Sweden (E-mail: goran.hansson{at}cmm.ki.se).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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