Published online before print June 28, 2007, doi: 10.1161/ATVBAHA.107.142810
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© 2007 American Heart Association, Inc.
Brief Reviews |
Murine Models of Vascular Thrombosis
From the Departments of Human Genetics (R.W.), Internal Medicine (M.W.), and the Division of Cardiology (D.E.), University of Michigan Medical Center, Ann Arbor.
Correspondence to Randal J. Westrick, University of Michigan, 5214 LSI Building, 210 Washtenaw Avenue, Ann Arbor, MI 48109. E-mail westricr{at}umich.edu
Series Editor: Daniel T. Eitzman
Regulation of Hemostasis and Thrombosis: Insights from Murine Models
ATVB In Focus
Previous Brief Reviews in this Series:
•Eitzman D. Regulation of hemostasis and thrombosis: insights from murine models. 2007;27:453.
•Tollefsen D. Heparin cofactor II modulates the response to vascular injury. 2007;27:454–460.
•Fay WP, Garg N, and Sunkar M. Vascular function of the plasminogen activation system. 2007;28:1231–1237.
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Abstract |
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Top
Abstract
Importance of Thrombosis in...Thrombotic complications of vascular disease are the leading cause of morbidity and mortality in most industrialized countries. Despite this, safe and effective drugs targeting these complications are limited, especially in the chronic setting. This is because of the complexity of thrombosis in both arteries and veins, which is becoming increasingly evident as numerous factors are now known to affect the fate of a forming thrombus. To fully characterize thrombus formation in these settings, in vivo models are necessary to study the various components and intricate interactions that are involved. Genetic manipulations in mice are greatly facilitating the dissection of relevant pro- and antithrombotic influences. Standardized models for the study of thrombosis in mice as well as evolving techniques that allow imaging of molecular events during thrombus formation are now available. This review will highlight some of the recent developments in the field of thrombosis using mouse models and how these studies are expanding our knowledge of thrombotic disease.
Thrombotic complications of vascular disease remain a leading cause of mortality worldwide. Effective chronic treatment strategies are limited in part because of an inadequate understanding of the factors that influence thrombosis. This review focuses on models of thrombosis useful for elucidating important regulators of thrombosis in mice.
Key Words: animal models • thrombosis • vascular biology • atherosclerosis • mouse strains
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Importance of Thrombosis in Various Disease States |
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The prevention of excessive blood loss after injuries that disrupt the vasculature is a critical survival response accomplished by the rapid and highly regulated formation of hemostatic plugs. This is a complex process that involves interactions between the vascular wall, platelets, leukocytes, coagulation proteins, and other as yet unidentified components. The deterioration of the hemostatic balance becomes apparent in aging organisms as they develop susceptibility to thrombotic events in both the arterial and venous vasculature.1 Thrombotic occlusion of major arteries supplying vital organs, such as the heart and brain, typically occur in the setting of preexisting atherosclerosis and are the leading cause of mortality in industrialized nations.2 Bacterial sepsis and other diseases are also known to trigger thrombosis.3 In these and other life threatening events involving thrombosis, there are both genetic and environmental contributors.4–6 Although several factors involved in these processes have been identified and targeted successfully with therapeutic interventions,7,8 there are clearly many that remain to be elucidated. Components that globally affect thrombus formation within distinct arterial or venous vascular beds exist; however, it is probable that there are vascular site-specific and disease-specific genetic regulators.9,10 Considering the widespread and critical role of thrombosis in many diseases, a more complete understanding of the processes involved in hemostasis and thrombosis may have important implications for treatment and prevention of common diseases such as myocardial infarction and stroke.
Because of the multifactorial nature of thrombosis and the difficulty in elucidating factors involved in human disease, there is a need for in vivo animal models to explore the pathophysiology of thrombosis. Mouse models of gene deletion or overexpression presently exist for virtually all known genes involved in hemostasis.11 It is currently possible to study variation in expression of single or multiple targeted genes in a variety of complex phenotypes in mice. The escalating availability of transgenic mouse models will soon enable the unprecedented capacity to explore the in vivo relevance of any gene of interest.
Since Virchow’s12 seminal investigations in dogs, scientists have been interested in the study of animal models of thrombosis, with a multitude of methods developed to produce thrombosis by vascular injury. Many of these earlier studies have been summarized periodically.13,14 These studies were often conducted in large mammals and have generally served as the basis for the development of mouse models, which have become increasingly used since the beginning of the transgenic mouse era. The purpose of this review is to describe some of the recently developed methodologies and their application to study thrombosis in mice.
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Intravital/Laser-Induced Vascular Thrombosis |
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Gross visual inspection of a forming clot was one of the earliest quantitation methods.12 Since that time, more sophisticated imaging methods have been developed that allow visualization of molecular events during thrombus formation (Table 1
). Arfors et al developed an early intravital microscopy procedure by implanting a transparent chamber on a rabbit ear which subsequently vascularized.15 They used a pulsed ruby laser to initiate thrombus development in the neovascularized chamber.14 Rosen et al developed a conceptually similar but noninvasive intravital mouse ear vein injury model using an argon-ion laser as the injury stimulus.16 Others have used fluorescently-loaded platelets to visualize platelet activation in mesenteric arterioles and venules.17,18 This system has been used to observe events that occur after stimulation with calcium ionophore18 or ferric chloride injury (see below).17 Falati et al also made use of fluorescent labeling to develop a confocal intravital microscopy system.19 This system can provide both brightfield images as well as images of the accumulation of fluorescently labeled antibodies specific for fibrin, CD41, tissue factor, or platelets. As such, multiple components of the growing thrombus can be simultaneously monitored, enabling a more precise characterization of the dynamics of clot formation (Figure 1C). Systems dependent on fluorescence are most effectively used when investigating thrombosis in small vessels, as the fluorophores used in these experiments emit in the visible spectrum, and are scattered by larger vessels.20
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Ferric Chloride Model of Arterial Thrombosis |
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The use of ferric chloride as a thrombogenic agent has become a common method for investigating the mechanisms of thrombosis in transgenic mice21 (Table 1
). In 1944, ferric chloride was first applied to the rabbit inferior vena cava to induce thrombosis.13 This method was adapted to rat carotid arteries by Kurz et al in 1990.22 They found that ferric chloride solution applied topically to the exposed rat carotid artery induced occlusive thrombosis, with the time to occlusion dependent on ferric chloride concentration. A thermocoupled temperature sensor was used to detect cessation in blood flow by measuring a decrease in target vessel temperature. Scanning electron microscopy of the occluded artery revealed evidence of endothelial damage associated with a clot composed of activated platelets, fibrin, and erythrocytes. These investigators suggested that the ferric chloride penetrated the vascular wall to precipitate thrombosis. This was shown in a recent study by Tseng et al in which topically applied ferric chloride traversed the endothelium by an endocytic-exocytic pathway into the arterial lumen to cause complete endothelial denudation and occlusive thrombosis.23 In 1998, the ferric chloride model was adapted to mice to study the effects of plasminogen activator inhibitor (PAI) 1 (Pai1) deficiency on arterial thrombolysis.24 Instead of a temperature sensor, a miniature ultrasonic flow probe (model 0.5VB, Transonic Systems) connected to a flowmeter (Transonic model T106) was placed around the vessel to measure blood flow in the isolated carotid artery. A single 1x2 millimeter strip of Whatman filter paper was soaked in a solution of 10% ferric chloride and applied to the adventitial surface of the carotid artery (Figure 1A). After 3 minutes, the paper was removed, the probe and artery were covered with saline, and flow monitoring was resumed until the end point of occlusive thrombosis was reached. In this study, it was also demonstrated that a smaller piece of paper with a higher ferric chloride concentration could be applied proximal to the flow probe to allow uninterrupted monitoring of blood flow.
Although this work did not reveal significant differences attributed to Pai1 deficiency in the time to occlusive thrombus formation, histological analysis of the arterial injury site revealed a noncircumferential full thickness vessel wall injury with an intraluminal platelet-rich thrombus. This thrombus was measured 24 hours postprocedurally and found to be reduced in Pai1-deficient mice.24 These results suggested that Pai1 deficiency enhanced fibrinolysis leading to increased vessel patency at 24 hours after injury, without marked effects on initial thrombus formation. Thus, the ferric chloride model was useful for the study of initial thrombus formation as well as for the study of factors involved in clot lysis.
Although 10% ferric chloride was effective in this study,24 it is possible that the concentration may have to be optimized for each pharmacological intervention, protocol variation, or mouse strain(s) under investigation. Recently, Wang and Xu performed a dose–response study to look at the effect of ferric chloride concentration on thrombus induction.25 These investigators found the threshold ferric chloride concentration to be 2.5% for producing occlusion at 10 minutes in C57BL6/J mice. They used heparin and clopidogrel to demonstrate that thrombosis induction at this concentration is more sensitive for the study of the anticoagulant and antiplatelet drugs compared with ferric chloride at higher concentrations.
In other iterations of the ferric chloride model, Smyth et al found that 20% ferric chloride dissolved in surgilube was effective in thrombus induction using a temperature-dependent thrombosis detection system.26 Schlacterman et al found 15% ferric chloride solution to be effective for determining effects of the factor V Leiden mutation on a hemophilic mouse strain of mixed C57BL6/J-129 genetic background.27
The ferric chloride model has also been applied to study thrombosis in other mouse blood vessels including the inferior vena cava28,29 and the microvasculature.17
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Systemic Collagen/Epinephrine-Induced Thrombosis |
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Intravenous injection of collagen and epinephrine into mice induces widespread platelet activation and subsequent mortality attributable to pulmonary thromboembolism.30 This model has been used to test the in vivo antithrombotic effects of pharmacological compounds and more recently to study the impact of genetically targeted platelet and other thrombotic factors in mice31 (Table 1
). There are many different types of collagen available from numerous vendors. Careful attention should be paid to obtaining the appropriate collagen and adhering to previously developed protocols to avoid lengthy model re-optimization times. |
Mechanical Injury Models |
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Several physical methods have been devised for the production of vascular thrombosis in animals.14 Mouse mechanical injury models developed primarily to look at vascular remodeling/intimal hyperplasia may serve as thrombosis models when analyzed acutely but may elicit a more variable thrombotic response. Methods previously used involve ligation,32 perivascular electrical injury,33 and placement of a polyethylene cuff.34 Ligation models that induce stasis have been particularly useful for studying factors involved in venous thrombosis.35
A wire injury model in which a 0.25- or 0.35-millimeter angioplasty guide wire is advanced into the femoral (Figure 1D) or carotid artery, respectively, has been commonly used to induce intimal hyperplasia in mice, which develops over the course of several weeks after injury.36,37 The wire is commonly passaged from 1 to 6 times and has also been passaged with rotation.37–40 Thrombus formation occurs in the acute injury period that may vary with genetic or pharmacological interventions.41 For example, when studying the neointimal response to injury in mice overexpressing a C-reactive protein human transgene, Danenberg et al noted persistent thrombus in mice expressing the transgene indicating that C-reactive protein may affect the thrombotic response to injury. This prothrombotic response was confirmed in the Rose Bengal carotid injury model.41
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Photochemical Model of Arterial Thrombosis |
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Reactive oxygen species are thought to be potent contributors to endothelial damage leading to atherosclerosis and thrombosis.42 The ability of the photosensitizing dye, Rose Bengal (tetrachlorotetraiodofluorescein), to produce reactive oxygen intermediates has been well documented.43 Thus, Rose Bengal–derived reactive oxygen species may be used to mimic the endogenous endothelial injury process and have been used effectively to induce focal vascular injury leading to thrombosis (Table 1
). Rose Bengal is particularly effective for in vivo studies because of its high photochemical efficiency and low systemic toxicity.43 It is typically injected or topically applied and is inert until exposed to 543-nm wavelength light. This light induces singlet oxygen radical formation at the target site. In 1985, Watson et al demonstrated the in vivo utility of Rose Bengal by showing that reproducible cerebral infarction in rats could be induced after intravenous injection of Rose Bengal and irradiation of the parietal convexity with 543-nm light from a filtered xenon arc lamp.44 A relatively simple procedure of thrombosis induction using Rose Bengal was used to investigate thrombosis in mice with genetic alterations in Pai1.45 This method applied the same flow monitoring system as used in the ferric chloride model to the surgically isolated common carotid artery.24 A portable 1.5-milliwatt green laser light system (540 nm; www.mellesgriot.com/) was then trained on the midpoint of the common carotid from a distance of 5 cm. This exposed approximately 1 mm of the carotid artery to photoactivating green light (Figure 1B). Rose Bengal, 50 mg/kg, was then injected via the tail vein and occlusive thrombosis was achieved approximately 50 minutes after the onset of injury in wild-type C57BL/6J mice. The injury to the endothelium is thought to occur as a result of reactive oxygen species generated when the green light encounters the circulating, or endothelial bound, Rose Bengal.46
This oxygen radical–induced injury may more closely approximate the mechanism of human arterial thrombosis compared with the ferric chloride model, as it closely mimics what is thought to occur in human arterial thrombosis.47 This could also constitute a milder form of injury as the time to occlusion is significantly longer in this model compared with ferric chloride at the doses commonly used. Thus, there is potential greater sensitivity for detecting alterations in factors affecting occlusive thrombosis. For example, the effect of Pai1 deficiency on time to occlusive thrombosis was highly significant in this model, suggesting that the fate of a forming thrombus may be affected by enhancing fibrinolysis.45
Based on the intensity of the light source, vascular target site, and mouse genetic background, Rose Bengal concentrations may also have to be optimized to elicit occlusive thrombosis, similar to the ferric chloride model.16,48–51 For example, when testing this model on mice of the KK/HlJ strain, a reduced concentration of Rose Bengal (37.5 mg/kg) had to be used to increase sensitivity to pharmacological interventions.52
The Rose Bengal model has also been used to study thrombosis in veins45,49 and in the microvasculature.16 Rosen et al compared the effects of anticoagulant and antiplatelet therapies on thrombus formation after the previously described intravital laser and a Rose Bengal-mediated injury to the mouse ear vasculature.16 After argon laser illumination with a short high-intensity pulse, thrombus formation was reduced after treatment with a GPIIb/IIIA antagonist but was not affected by the anticoagulants hirulog, PPACK, or NapC2. In contrast, thrombus formation after photochemical injury with Rose Bengal was inhibited by both anticoagulant and antiplatelet compounds. Thus, in this study, the Rose Bengal model was more sensitive to diverse pharmacological interventions than the laser model.
In the Rose Bengal carotid injury model, many investigators use time to sustained absence of blood flow as the primary end point. However, we have noticed that before achieving sustained occlusion, multiple episodes of clot formation and embolization or lysis are observed. Additionally, Matsuno et al documented cyclic lysis/embolization patterns in arteries but not veins of wild-type mice.49 These cyclic flow variations were also seen by Konstantinides et al using the ferric chloride model. They showed unstable flow patterns in vitronectin and Pai1-deficient mice compared with wild-type, suggesting thrombus instability attributable to increased thrombolytic activity.53 Tracking and analyzing these events may provide physiologically meaningful data, in addition to the end point of occlusive thrombus formation.
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Plaque Injury Models |
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Arterial thrombosis leading to myocardial infarction and stroke commonly occurs in the setting of preexisting atherosclerotic vascular disease.54 This suggests that the atherosclerotic plaque is necessary to incite occlusive macrovascular arterial thrombosis. In humans, the plaque grows into the arterial lumen and it is this aspect that becomes disrupted immediately preceeding myocardial infarction and stroke, either by plaque erosion or rupture. Subsequent exposure to plaque-related thrombogenic factors seems the likely initiator of these thrombotic events.54
Mouse models of arterial thrombosis that involve injury in the setting of preexisting atherosclerosis may be necessary to delineate the full complexity of the thrombotic response in this context. Mice deficient in apolipoprotein E or the low-density lipoprotein receptor have been widely used to study factors affecting atherogenesis. These mice develop atherosclerosis in a site-specific pattern with predilection for arterial branch points.55,56 For example, early reproducible lesions occur in the brachiocephalic trunk and later in the distal common carotid artery proximal and including the bifurcation into the internal and external carotid arteries.
To address the complex nature of thrombus development at the site of an atherosclerotic plaque, a number of plaque rupture or plaque injury models have been described (Table 2). In particular, the Rose Bengal photochemical model is suitable for causing injury to atherosclerotic plaques. This is technically possible because surgically exposed mouse arteries are translucent, and atherosclerotic lesions can be readily visualized through a dissecting microscope. Injury can thus be targeted to sites of atherosclerosis. This injury model has been applied to aged apolipoprotein E–deficient mice, and occlusive thrombosis formation at the site of the atherosclerotic plaque was demonstrated.57 Twenty-four hours after injury, plaque-associated thrombus persists (Figure 2), whereas thrombus in arteries without plaque have typically resolved. Thus, this model can be used to test interventions affecting thrombus formation and dissolution in the setting of an atherosclerotic plaque. For example, heterozygous tissue factor pathway inhibitor deficiency leads to a shortening of the time to occlusive thrombus formation after plaque injury, whereas deficiency of Pai1 is capable of prolonging it.51,58
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A number of plaque injury models have been developed in the context of hyperlipidemia, which sensitizes mice to atherosclerotic plaque development. An important consideration when using these models to test modulations of gene expression using noninducible transgenic or knockout mice bred to a hyperlipidemic background is that the genetic manipulation is present from birth and may affect the size or composition of the atherosclerotic plaque. A change in plaque growth or characteristics could affect thrombosis time, making it difficult to determine whether the gene affected the thrombotic response, plaque composition, or both.
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Spontaneous Thrombosis |
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Although myocardial infarction and stroke occur after occlusive thrombosis that develops at the site of a disrupted atherosclerotic plaque, mouse models that exhibit spontaneous vascular thrombosis can be effective in identifying candidate gene interactions, especially when a clear phenotype, such as mortality, is present. Mice homozygous for the factor V Leiden mutation experience strain-related spontaneous thrombosis but on the C57BL/6J strain they are born at the expected rate and demonstrate normal survival.59 Lethal perinatal thrombosis has been demonstrated to occur in this strain when other candidate prothrombotic genetic alterations are combined with the factor V Leiden allele. For example, the prothrombotic phenotypes of both protein Z deficiency and heterozygous deficiency of tissue factor pathway inhibitor were revealed when these mutations were combined with factor V Leiden.60,61 The primary end point of these studies was a marked reduction in live births of mice inheriting certain combinations of factor V Leiden and protein Z or tissue factor pathway inhibitor deficiency. This mortality was shown to occur in the perinatal period and was consistent with disseminated thrombosis. The rare survivors developed evidence of spontaneous arterial thrombosis with age.61
The perinatal mortality that occurs in mice with homozygosity for factor V Leiden and heterozygous tissue factor pathway inhibitor deficiency is currently being used as part of a genome wide screen designed to identify genes capable of rescuing perinatal mortality attributable to disseminated thrombosis.11 Thus, mouse strains such as those carrying the factor V Leiden mutation or other strains exhibiting lethality may have utility in genetic studies of thrombosis risk when used as a sensitizing strain,62 especially as complete sequence data from multiple strains becomes available. Strain-dependent differences in thrombotic responses may also be used to identify important novel genes.63 The development and use of multiple recombinant inbred lines and chromosomal substitution strains are already underway to map strain modifiers of hemostasis.64,65
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Vascular Site–Dependence of Thrombosis |
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Thrombus formation after injury may vary depending on the size of the blood vessel. For example, myocardial infarction and stroke are the result of occlusive thrombus formation in a relatively large artery. Many factors differ between macro- and microvessels that could affect thrombosis including hemodynamics,66 endothelial expression patterns, and vascular smooth muscle cell (VSMC) contribution.62,67 Different interventions may produce different results depending on the size of vessel injured. For example, whereas circulating tissue factor affected thrombus formation in the cremasteric arteriole model,68 it did not affect time to occlusion after injury to the carotid artery.69 Vessel wall tissue factor may be even more potent in the setting of a lipid-rich atheroma, where tissue factor is abundant.70
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Reproducibility of Thrombosis Models |
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Consistent times to occlusion in an inbred mouse strain are a prerequisite to analysis of a genetic or pharmacological intervention. For most of the models discussed, there is a learning curve that may involve weeks to months of practice before acceptable standard deviations in thrombosis times are achieved. There are also many other variables which could influence the outcome of thrombosis and should be carefully considered when undertaking studies using a thrombosis model.
Depth of anesthesia has been shown to be an important determinant of thrombosis times. Wilson et al71 found that after pentobarbital anesthesia in the nonventilated mouse, respiratory depression with carbon dioxide retention and acidosis occurs. These changes increase carotid blood flow and produce prolonged occlusion times. Wilson and Hatchell found that exogenous oxygen administration in the Rose Bengal model decreases time to occlusive thrombosis43 in a rat retinal vein thrombosis model. Thus, careful attention to respiratory status is required with consideration of mechanical ventilation if the anesthesia protocol leads to respiratory depression. Maintenance of physiological body temperature under anesthesia may also be important.
A number of transgenic mice display phenotypic variability in thrombosis severity depending on the strain background (Table 3). For example, we have found that heterozygous protein C–deficient mice have reduced survival on a mixed 129S1/SvImJ- C57BL/6J compared with a pure C57BL/6J background. This represents a particularly potent gene-strain background interaction. Mouse genetic background may also have potent effects on vascular response to injury. When tested using the Rose Bengal model, purebred C57BL/6J mice form occlusion times that are significantly longer than purebred mice of the KK/HlJ strain.52 Similar strain differences are also present between C57BL/6J, 129S1/SvImJ and other strains (Table 4). Because gene targeting is most often carried out in 129 strain-derived ES cells, and the resulting mice carrying the targeted mutation are commonly bred to the C57BL/6J mouse strain, a hybrid F1 results. Interbreeding F1 carriers can then result in a mouse homozygous for the targeted allele, as well as randomly inheriting any combination of the parental alleles at any given locus. These hybrids may confound results of knock-out studies.
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The study of heparin cofactor II (HcII)–deficient mice demonstrates the effect of homogeneous versus heterogeneous strain background in assessing the effects of a gene-targeted intervention on thrombosis. He et al performed the Rose Bengal thrombosis model on HcII-deficient mice on a mixed C57BL/6J-129/SvJ background.72 They were found to form occlusive thrombi in 27±5 minutes. After backcrossing 6 generations to the C57BL/6J strain, the average time to occlusion increased to 39 minutes.72 The increase in average time to occlusion on the HcII backcrossed mice was presumably attributable to the diminution of 129 strain alleles after the extensive backcrossing. In this case, despite the strain differences, the effect of the HcII knockout compared with wild-type was still apparent in both sets of experiments because the appropriate controls were used.
The appropriate controls to use for gene targeted hybrid mice are littermates. Once viable homozygotes are obtained, it is tempting to crossbreed homozygote with homozygote and wild-type with wild-type mice from the F2 generation to more quickly generate wild-type and homozygote experimental groups. This strategy should not be undertaken because different random sets of alleles from each parental strain will become fixed in each population, essentially resulting in the creation of 2 substrains with distinct genetic backgrounds. This could lead to spurious results misattributed to the gene of interest, even after extensive backcrossing.
Strain-related genetic differences often persist in the region nearby the targeted locus, a region called the differential chromosomal segment.73,74 Even after 10 generations of backcrossing, the congenic fragment can be over 20 megabases in size and represents the most significant portion of the remaining gene targeted strain DNA (www.informatics.jax.org/silver).73 Thus there is the possibility that phenotypic differences are attributable to a closely linked gene, rather than the targeted locus.73,74 This may be particularly relevant when investigating a knockout in a gene cluster, where many genes in the region have related function. The size of the differential chromosomal segment can be greatly reduced by speed congenics.75
The age and gender of the experimental mice should also be taken into consideration before beginning thrombosis studies. Age is clearly a risk factor for thrombosis in humans,1 but the effects of age have not been extensively studied in mouse thrombosis models. Takeshita et al noted age-dependent increases in Pai1 expression and fibrin deposition in the Klotho mouse, a murine model of aging.76 Yamamoto et al found pronounced increases in tissue factor and Pai1 gene expression in aged mice subjected to restraint stress.77,78 By quantitating fibrin deposition and intravascular thrombosis, we have found that the factor V Leiden mouse has also been useful to elucidate prothrombotic phenotypes in mice that develop age-related vascular disease.52,59,79 In addition, Bodary et al observed a significant increase in thrombotic tendency with age and fatness in mice of the KK/HlJ strain.52
Gender differences related to thrombosis have not been well documented in animal models to date, but there are clear gender effects in partial thromboplastin time, prothrombin time, and platelet count as outlined by the mouse phenome database (http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home).80 Based on these data, it is probable that there are gender effects on thrombosis in several mouse strains. It should also be noted that pregnancy can play a major role in thrombosis susceptibility. Fry et al reported spontaneous thrombosis in the left atria of BALB/c female mice, with disease penetrance increasing from 2.6% in virgin females to more than 50% in females after one or more pregnancies.81
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Future Considerations |
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With the completion of the human, mouse and other mammalian genome sequences, a major challenge in basic thrombosis and vascular research is to translate this sequence information into functional data, which can then be used to aid in the design of rational therapies. Mouse transgenic technology has already enabled researchers to develop many mutants with which to investigate the processes of vascular injury and thrombosis in vivo, with an existing knockout mouse model for virtually all known hemostatic genes.11 However, in the next few years there will be an explosion of genetic tools to further facilitate these investigations. For example, the arduous task of gene targeting can now be avoided with the development of trans-NIH mouse initiatives such as the KOMP project (http://www. nih. gov/ science/ models/ mouse/ index. html). This project aims to generate inducible knockouts for the entire spectrum of genes in the genome. The use of these mice will enable unprecedented opportunities to investigate the tissue specific expression of any gene of interest as it relates to thrombotic disease. It is currently possible for an individual to nominate up to 10 genes of interest for priority through the Jackson Laboratories website: http://www.informatics.jax.org/ mgihome/ KOMP/ nominationForm. shtml.
To accomplish this ambitious goal, the KOMP project will produce mouse lines with part or all of each gene flanked by loxP sites. By subsequent breeding to a transgenic mouse expressing the Cre recombinase (Cre) gene in the tissue or cell type of interest, tissue-specific deletion of a gene of interest may occur. Cre accomplishes this by recognizing and recombining sequences flanked by loxP sites. Many transgenic mice with Cre targeted to specific tissues or cell types have been generated. Information about the various Cre transgenic mice can be obtained through the Nagy laboratory website: http://www.mshri.on.ca/nagy/. When using Cre transgenic mice to generate tissue specific gene deletions, care should be taken in designing the mating strategy because germline recombination has been documented with the use of certain Cre transgenic mice.82,83 In this case, the maternal effect on recombination can be circumvented by using only male Cre transgenics in the mating strategy. For further information and discussions about Cre specificity, refer to the MGI email List Service archives: http://www.informatics.jax.org/mgihome/lists/lists.shtml.
In addition to the KOMP project, the ENU mutagenesis community is providing resources to create entire series of mutant alleles in any gene of interest. The chemical mutagen ENU (ethylnitrosourea) is used to create random point mutants in germ cells of male mice. Parallel DNA/sperm archives containing random mutants have been made that saturate the entire genome with point mutants84,85 (http://www.ingenium-ag.com/), enabling researchers to screen a series of random point mutant alleles, called an allelic series, in any gene of interest.86,87 Many of these alleles will have mutations in functional domains, thus allowing protein function to be thoroughly investigated. These point mutants may also prove to more closely approximate human conditions than traditional mouse transgenics, as 70% of all disease causing alleles so far described in humans are point mutants.85
Another resource now being offered that may speed the discovery of novel therapeutics effective at modulating blood coagulation is provided through the Molecular Libraries Roadmap initiative (http://nihroadmap.nih.gov/molecularlibraries/), which makes small molecule libraries publicly available for high throughput screening. The identification of candidate therapeutic molecules generated via high throughput thrombosis screens will need to be validated using mammalian models.
Organization and recordkeeping of the increasing numbers of mouse models generated by the above approaches and used by any single laboratory can be made easier by the use of excellent mouse colony management software freely available from and supported by the Jackson laboratory (http://www.jax.org/jcms/index.html).
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Conclusion |
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Current pharmacotherapy for preventing the most common cause of mortality, atherothrombosis, is limited at present. The progress of the past few years in developing and adapting mouse models of thrombosis and vascular disease has positioned the field to make full use of the vast genetic and molecular resources that are becoming available. Continual refinement and use of mouse vascular models will progressively broaden our understanding of the mechanisms underlying thrombus formation. The knowledge gained may be applied toward many physiological and pathological processes in which thrombosis plays a role.3,88 Investigators have already used several antithrombotic compounds to validate these models and are now using mouse models to generate preclinical information about candidate compounds under development (Table 5). Information generated from these studies will eventually translate to safe and effective therapies for thrombotic disease.
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Acknowledgments |
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We gratefully acknowledge Peter Gross for the Figure 1C image showing the laser induced thrombosis.
Sources of Funding
This work was supported by funds from the National Heart, Lung, and Blood Institute (HL057346; to D.E.) and an American Heart Association predoctoral fellowship (0315212Z; to R.W.).
Disclosures
None.
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Footnotes |
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Original received February 22, 2007; final version accepted June 7, 2007.
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References |
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