Cardiovascular Research 39 (1998) 8–33
Review
Mouse models of angiogenesis, arterial stenosis, atherosclerosis and
hemostasis
´ ´ Collen
Peter Carmeliet*, Lieve Moons, Desire
Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Leuven, B-3000, Belgium
Received 2 March 1998; accepted 13 March 1998
Abstract
The development of efficient transgenic technologies in mice has allowed the study of the consequences of genetic alterations on
cardiovascular (patho)physiology, although the development of such models have been hampered by size limitation of species resulting in
time-consuming, labor-intensive and costly analyses. This overview summarizes the murine models currently available for studying or
manipulating angiogenesis, arterial stenosis, atherosclerosis, transplant arteriopathy, thrombosis, thrombolysis and bleeding and addresses
techniques to evaluate vascular development during embryogenesis.  1998 Elsevier Science B.V. All rights reserved.
Keywords: Angiogenesis; Atherosclerosis; Restenosis; Bleeding; Thrombosis; Transplantation; Adenovirus
1. Blood vessel formation
Blood vessels are among the first organs to develop
during embryogenesis [1]. Vascular development proceeds
in the mouse during the early postnatal period in the lung
[2], the heart [3], the brain [4,5] and the retina [6]. During
adulthood, blood vessel formation resumes during reproduction and wound healing. Excess formation of blood
vessels may contribute to hypertrophic scar proliferation,
chronic inflammatory disorders, diabetic retinopathy, or
tumor growth [7]. On the contrary, insufficient blood
vessel formation is responsible for tissue ischemia, delayed
wound healing and chronic ulcers. Blood vessels initially
develop as endothelial cell-lined channels by in situ
differentiation of angioblasts and their subsequent assembly into a primitive vascular plexus (vasculogenesis) (Fig.
1a). Subsequently, the primitive vascular network expands
and remodels via sprouting angiogenesis (budding of new
sprouts from preexisting vessels), nonsprouting angiogenesis (intercalated growth of endothelial cells) or intussusceptive growth (whereby new intercapillary pillars split
preexisting vessels). Investment of the endothelial tubes by
*Corresponding author. Tel.: 132 (16) 345 772; Fax: 132 (16) 345
990; E-mail: [email protected]
0008-6363 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 98 )00108-4
periendothelial mural cells (pericytes around capillaries,
smooth-muscle cells around arteries) is essential for their
structural integrity and vasomotor control [8–10] (Fig. 1b,
c). Increased vascular supply can also be mediated by
remodeling of preexisting vessels such as of the coronary
arteries [11]. As recent discoveries have identified several
candidate angiogenic or angiostatic factors, increasing
interest has arisen to study their biological role or to test
their potential therapeutic value. Consequently, the need
for quantitative angiogenesis assays has been growing
during recent years. Whereas previous reviews have discussed the use of angiogenesis assays in other species
[12–14], this section will review the use of the mouse as a
model to study blood vessel formation and function.
1.1. Vascular development in the mouse embryo
The study of the embryonic vasculature has yielded
novel insights in the molecular mechanisms of blood
vessel formation [1,8,10]. Histological analysis, immunostaining and in situ hybridization for endothelial, periendothelial or basement membrane markers allows the study
of embryonic vessels on tissue sections or in whole mount
Time for primary review 10 days.
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
9
Fig. 1. Different phases of embryonic vascular development. (a) Endothelial precursors (angioblasts) differentiate to early endothelial cells, which become
assembled into a primitive capillary plexus (vasculogenesis). This emerging network expands via intussusceptive growth, intercalated growth and sprouting
angiogenesis, after which it becomes remodeled via pruning, fusion and regression of preexisting vessels into a tree of arteries, capillaries and veins.
Endothelial cells further differentiate and acquire specific properties such as the formation of a tight barrier in the brain, or the formation of fenestrations in
exocrine glands. (b) Mesenchymal cells differentiate to pericytes (PC) and primitive smooth-muscle cells (SMC), which become recruited around the
endothelial tubes. Mural cells migrate longitudinally alongside the endothelial tubes or sprout from preexisting muscularized vessels. Both endothelial and
mural cells constitute the essential cellular components of a mature vascular network.
embryos [15–17]. Specific markers can be used to identify
the different vascular beds; for example, GLUT-1 marks
the developing blood–brain barrier [18], whereas FLT-4
identifies lymphatic vessels (at least beyond midgestation)
[19]. The embryonic vasculature can also be visualized by
inter-crossing the target mice with other transgenic mouse
strains, expressing a ß-galactosidase marker gene in endothelial or mural cells [20–23] (Fig. 2a, b). Computerassisted image analysis can be used to reconstruct the
three-dimensional pattern of the embryonic vasculature
[15]. In addition, barium-gelatin angiography, and scanning electron or light microscopy of vascular casts in
embryos aged 9 to 20 days have been used to visualize
their three-dimensional architecture [2]. Blood vessel
function and blood flow can be studied in embryos, freshly
dissected and still attached to their yolk sac during the
early stages of embryogenesis (,12.5 days of gestation),
or in cultured embryos during the initial period of vascular
development (8.0 to 10.5 days of gestation) by transillumi-
nation of the transparent yolk sac and embryo [24,25], or
after injection of fluorescent dyes (microangiography) [26]
or other tracers (ink) [27] (Fig. 2c, d). Injection of trypan
blue or horse radish peroxidase has been used to study the
development of the blood brain barrier [18]. Pulsed
Doppler has been used to measure blood velocity in 10.5to 14.5-day-old embryos in utero while maintaining
uteroplacental continuity [28]. Noninvasive high-resolution
nuclear magnetic resonance imaging may provide future
opportunities to monitor vascular development.
The developing embryonic vasculature (like other
neovessels) is sensitive to (anti)-angiogenic factors. Immunoglobulins (type IgG) and only a few growth factors
(including TGF-ß1) will be transferred across the yolk sac
or placental barrier after intravenous injection into the
maternal plasma of pregnant females or upon supplementation into the medium of cultured embryos [29,30].
Other molecules (growth factors, antibodies, or drugs) with
(anti)-angiogenic properties have to be injected into the
10
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
Fig. 2. Angiogenesis in the mouse embryo. (a) Whole mount staining for b-galactosidase of a wild-type 9.5 day-old embryo expressing the blue marker in
the entire vasculature after interbreeding onto a TIE1:LacZ background. (b) Section through a 9.5 day-old embryo (the same as in panel a), revealing the
blue endothelial cells throughout the entire vasculature. (c) Ink injection into the heart of a 9.5 day-old embryo, visualizing the embryonic vascular
network. (d) Whole mount view of a cultured 9.5 day-old embryo (still attached to the transparent yolk sac), revealing the blood-filled vitelloembryonic
vessels.
Fig. 6. Atherosclerotic aneurysm formation in the mouse. (a–c) Histological sections through the aorta from an apoE-deficient mouse (fed a cholesterol-rich
diet), revealing fragmentation of the elastic laminae (a), thinning of the media and dilatation of the aortic wall (b), rupture of the aneurysm (c). (d) Loss of
medial a-actin immunoreactive smooth-muscle cells associated with media destruction.
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
extracoelomic cavity or, directly, into the heart of the
embryo in utero or in culture [25]. Intracardiac injection of
recombinant adenoviruses, resulting in the preferential
expression of the transferred genes in the embryonic
vascular endothelium, allows visualization (ß-galactosidase
marker gene) or genetic manipulation (anti-angiogenic
genes) of the embryonic vasculature [26,31].
1.2. Vascular development in the neonatal mouse
Physiological development of blood vessels in the
mouse proceeds during the first postnatal weeks. As
growing and remodeling blood vessels are more sensitive
to modulation by (anti)-angiogenic factors than mature,
quiescent vessels, and since vascular growth in the adult
mouse is restricted to reproduction or to pathological
conditions (tumorigenesis, inflammation etc.), these
neonatal angiogenesis models offer a unique opportunity to
study the molecular mechanisms of physiological vessel
formation.
The myocardium becomes vascularized during embryonic development by an interconnected network of
coronary arteries, capillaries and veins [32]. Immediately
after birth, the pulmonary pressure drops (as a result of
lung inflation), concommittant with a rise in the systemic
pressure. The heart adapts to these hemodynamic changes
by a two- to three-fold hypertrophy (and, to a limited
extent, hyperplasia) of the cardiomyocytes [3]. The increased metabolic demands of the postnatal heart are met
by a significant angiogenic response during the first two
postnatal weeks, resulting in a two-fold increase in the
number of myocardial capillaries. Methods for morphometric quantitation of the capillary density, the capillary-to-cardiomyocyte fibre ratio, the capillary length,
surface or volume densities and proliferation have been
described [3]. The tree of larger coronary vessels, which
are mature at birth, can be visualized by corrosion casting
or microangiography [33].
The retina is largely avascular at birth, and only
becomes vascularized during the first three weeks of life.
Natural development of the retinal vasculature is considered to be regulated by oxygen and mediated by vascular
endothelial growth factor (VEGF) [6]. Physiological levels
of hypoxia, caused by increasing demands for oxygen at
the onset of neuronal activity, are detected by strategically
located populations of neuroglia that secrete VEGF and
induce the formation of retinal vessels. As the vessels
become patent, the hypoxic stimulus is relieved, so vessel
formation is matched to oxygen demand. Vessel regression
is a natural response to oxygen surplus, resulting in
capillary remodeling and formation of capillary-free zones
[34]. Initially, a capillary plexus consisting of endothelial
cell-lined channels is formed, which subsequently becomes
invested by pericytes. The retina is particularly suited to
study pericyte biology as it contains the highest ratio of
pericytes versus endothelial cells [35]. The retinal vascula-
11
ture is readily accessible for qualitative and quantitative
analysis via angiograms using fluorescent markers, whole
mount staining (using endothelial or pericyte-specific
markers), or histological analysis [36,37]. As the immature
retinal vasculature is more sensitive to changes in oxygen
concentrations than the mature adult network, a model of
ischemic retinopathy was developed whereby hyperoxia
induces irreversible damage to immature retinal vessels of
the neonate, resulting in intense retinal ischemia [37].
When the neonate is returned to normoxia, a second phase
is initiated, distinguished by excessive revascularization
with abnormally leaky vessels. This model, which mimicks
to a certain extent the vascular response during retinopathy
of prematurity or diabetic retinopathy, may be useful to
test the efficacy of (anti)-angiogenic molecules [38].
Another vascular network in the eye which undergoes
significant remodeling shortly after birth is the hyaloid
vasculature, which grows into the vitreum and onto the
surface of the lens [39,40]. The hyaloid system consists of
the tunica vasculosa lentis (on the posterior lens), the vasa
hyaloidia propria (in the vitreum), and the pupillary
membrane (on the anterior surface of the lens in the
anterior chamber). Regression of this system, which disposes cells that would disturb the passage of light to the
retina, begins with the vasa hyaloidia propria at about 5
days after birth and is completed by about 21 days. The
pupillary membrane has proven useful for analysis, mainly
because it can be removed from the eye by dissection and
visualized in toto. Vital and histological analyses can be
used to determine the blood flow through and the fate of
whole capillary segments in vivo [40].
The growth and development of the brain and its blood
vessels are intimately linked [5]. The somatosensory cortex
is an attractive model for following this development
because its topographic neuronal organization and its
integrated vasculature are largely absent at birth and
develop postnatally. Neonatal mice have dense surface
capillary networks with numerous anastomoses between
arterioles and relatively small and irregular veins. Intraparenchymal blood vessels grow from endothelial sprouts that penetrate the embryonic brain from the pial
vascular plexus. Morphological changes over the first two
postnatal weeks include a three- to four-fold increase of
the intraparenchymal capillary density, and an associated
increase in the intraparenchymal capillary length (likely
reflecting an adaptation to increased neural activity and
metabolic rate). These angiogenic changes appear to be
mediated in part by VEGF [4]. In addition, a reduction in
the density of the pial vascular network occurs, which
contains venules with increased diameter, length and less
tortuous trajectory, and fewer arteriolar anastomoses. In
vivo videomicroscopy with fluorescent tracers in cranial
windows has been used to image the dynamic changes in
patterning and blood flow in anesthetized mice [5]. Perfusion with photographic emulsion allows reconstruction of
cortical blood vessels, whereas horse radish peroxidase
12
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
injection has been used for the measurement of permeability [5].
The rate of postnatal growth of the lung is most rapid in
the neonatal period, resulting in a 10-fold increase in the
number of alveoli, and a doubling in the alveolar size [41].
At birth, the distribution of pre-acinar arteries is completed, but with increasing age, there is a significant
growth of many small intra-acinar arteries as the lung
adapts from a high-pressure / low-flow system to one with
low pressure / high flow. Structural remodeling of the walls
of pre- and intra-acinar arteries and veins is a striking
feature of normal lung growth. External (pathological)
stimuli to the lung and its vessels (including changes in
oxygen or blood pressure) during this period of rapid
expansion induce responses greater in magnitude than
stimuli occurring after growth has ceased.
1.3. Angiogenesis in the adult mouse
Neovascularization in the adult mouse results in most
instances from angiogenic sprouting of new capillaries
from preexisting vessels [8], although colonization of
peripheral tissues by bone marrow-derived angioblasts has
recently been proposed as an alternative mechanism [42].
In addition, remodeling of larger preexisting vessels (such
as of the coronary arteries) is an alternative mechanism
whereby the increased demands for vascular supply of
nutrients and oxygen are met [11]. Lymphangiogenesis is a
poorly characterized process that only recently has become
accessible to molecular analysis [43]. The following
discussion reviews the different characteristics of assays,
categorized according to their use in studying angiogenesis
induced by (i) application of angiogenic factors; (ii)
implantation of tumors; (iii) wound healing; (iv) tissue
ischemia; or (v) lymphangiogenesis (Fig. 3).
The effect of (anti)-angiogenic molecules has been
frequently studied in the corneal micropocket assay [44].
Implantation of hydron pellets containing angiogenic factors in the stroma of the avascular mouse cornea allows
screening of the angiogenic response in the absence of
inflammation. As the cornea in the mouse is thinner than in
other species, growth of new blood vessels largely occurs
in a two-dimensional plane. Implantation of pellets on the
iris into the anterior chamber of the eye is technically more
challenging due to its small size. This model (which can
also be used to study vascularization induced by implantation of tumor cells, or by thermal or chemical injury) is
strain-dependent, as nude mice, spontaneously develop a
higher degree of vascular channels within the peripheral
stroma [45]. Other mouse strains, including Corn1 mice,
spontaneously develop irregular thickening of the corneal
epithelium and significant stromal neovascularization [46].
Chronic transparent chambers in the brain and the dorsal
skin offer the advantage of quantitating the length, surface
area, volume and number of vessels of the network, as well
as the dynamic changes of blood flow, permeability, and
shear stress using videomicroscopy and computer-assisted
image analysis [47,48]. The skin model allows visualization of blood vessels by transillumination, whereas epiillumination and injection of contrast agents (fluorescent
dyes, photographic emulsion, corrosion casts) are required
in the cranial model.
An alternative method to study new vessel growth
involves the implantation of a polymer matrix (gel,
sponge, capsules etc) containing (anti)-angiogenic factors
[49–52]. Although analysis of blood vessel formation in
the latter model largely relies on histological analysis,
insights in the angiogenic response can be obtained
through quantitation of the hemoglobin and red blood cell
counts, measurements of blood-flow rates using radioactive
Fig. 3. Overview of the different angiogenesis models in the mouse.
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
tracers, or analysis of biochemical parameters, characteristic of angiogenesis (such as for example extracellular
matrix turnover).
In contrast to the significant angiogenic response in
other rodents [53], neovascularization in the murine
peritoneum after intraperitoneal injection of angiogenic
factors is minimal (V. Laux, personal communication).
However, because of their transparency (|120 mm thin),
the exteriorized peritoneum or cremaster muscle offer the
advantage of evaluating in situ, the microvascular changes
in permeability, flow-rates, vasomotor activity and
leukocyte rolling after topical application of angiogenic
factors [54–56].
The hairless mouse, although similar in appearance to
the nude mouse, is immunologically competent, with a
normally functioning thymus and T-cells [57,58]. The ear
of the hairless mouse comprises a central cartilaginous
sheet, sandwiched between two full-thickness dermal
layers (together |300 mm thick), and measures 10 to 13
mm in width and length. Its nourishment is supplied by
three to four neurovascular bundles, which can be directly
observed through vital microscopy. Neovascularization
after injection of angiogenic factors (or implantation of
tumors, or full thickness wounds) can be quantitatively
evaluated [59], although the model suffers from the limited
ability to keep the grafted tissue undisturbed and from the
poor optical quality after graft implantation.
Tumor vascularization, and its modulation by (anti)angiogenic factors [60], has been studied using chronic
transparent chambers in the brain and the skin [47,48].
Videomicroscopy of the vascularization of tumors implanted in the chambers allows easier visualization than in
other tissues without a chamber [61]. Xenografts can be
implanted in chambers placed in immunodeficient recipients. Ingrowth of human vessels into human tumor grafts
can be studied by prior transplantation of full-thickness
human foreskin grafts onto immunodeficient mice, and
subsequent grafting of human cancer cells into the human
skin [62]. However, after 3 to 4 weeks, murine blood
vessels progressively vascularize the human tumor. Matrigel, a reconstituted extract of basement membrane,
induces an angiogenic response, enhancing thereby the
growth of tumors which cannot be grown in xenogenic
immunodepressed hosts, even when inoculated at high cell
doses [63]. Agarose microencapsulation of tumor cells,
attached to microcarrier beads, has been used to isolate the
tumors from the immune system of nonsyngenic hosts
[64]. Tumor vascularization can also be evaluated by
implantation of the tumor cells in the stroma of the mouse
cornea, but the angiogenic response is confounded by
inflammation. As the role of the (orthotropic) microenvironment of the host tissue is becoming increasingly
recognized, other tissues such as the mouse liver have been
used to study tumor neovascularization [65].
In contrast to the angiogenic response induced by
tumors implanted into (immunodeficient) hosts, vasculari-
13
zation can be also studied in tumors spontaneously developing in situ in transgenic mice carrying dominant
oncogenes (bovine papilloma virus-1 genome [66], Simian
virus 40 large-T antigen [67], Fps / Fes tyrosine kinase
[68], Polyoma middle-T antigen [69,70]) or in tumor
suppressor-knockout mice (p53) [71]. Such transgenic
models more closely mimics the multistage growth of
carcinogenesis with the onset of an angiogenic switch as a
hallmark of malignant tumor progression. In addition, they
allow three-dimensional tumor growth, required for reproduction of (hypoxic / hypoglycemic) stress-induced
tumor angiogenesis [72,73]. Unfortunately, direct visualization of the dynamic aspects of the microcirculation via
microvascular techniques is limited. Noninvasive highresolution nuclear magnetic resonance imaging may provide a means for monitoring the development of the tumor
vascularization over its entire time course [72].
Vascularization of healing wounds is a natural process,
which can be studied using the corneal chemical or thermal
injury model, the dorsal skin or cranial window models,
the hairless mouse ear model, or the models relying on
implantation of gels, sponges or perforated capsules,
containing (anti)-angiogenic factors [74].
Models of angiogenesis, induced by tissue ischemia,
have been recently developed in the mouse. Limb ischemia
due to permanent ligation of the femoral artery, induces the
formation of collateral vessels over a time course of
several weeks [75]. Revascularization of the ischemic
extremities can be monitored noninvasively using Laser
Doppler perfusion imaging, which has also been used to
measure microregional erythrocyte flux in tumor implants
in mice [76]. As synchrotron radiation microangiography
(with a resolution limit of 30 mm) has been recently used
to visualize small collateral arteries in the rat [77], it may
provide a means to detect changes in the microcirculation
in the mouse as well. Permanent ligation of the left anterior
descending coronary artery in the murine heart induces
myocardial ischemia, resulting in the infiltration of the
ischemic regions by a highly vascularized granulation
tissue as well as in the remodeling of the coronary arteries
( [33] and unpublished observations). Microangiography
and corrosion casts have been used to visualize the
myocardial vascular network [33]. A mouse model of
diabetic retinopathy based on the prolonged feeding of
galactose-rich diets has been developed [78]. Although this
model is characterized by the typical saccular microaneurysms, thickening of the capillary basement membrane and pericyte drop-out, neovascularization does not
occur in this model. The model of ischemic retinopathy in
neonates [37] has been described above.
Fluorescence microlymphangiography allows the examination of the endogenous or tumor-associated lymph
circulatory system in the mouse [79,80]. Although lymph
capillaries can be histologically distinguished from vascular channels by their underdevelopment of a basement
membrane, absence of red blood cells (except during
14
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
conditions of injury or infection), and lack of tight
junctions, the identification of a novel lymph-angiogenic
marker, FLT-4 [19], and the generation of mice expressing
the ß-galactosidase marker under control of the endogenous FLT-4 promoter (K. Alitalo, personal communication)
might aid in the visualization of these lymphatic structures.
1.4. Gene transfer in the vasculature
Gene transfer offers the opportunity to genetically
manipulate the endothelium (smooth-muscle cell gene
transfer in the mouse is discussed in the section on arterial
restenosis). Due to its small size, only systemic instead of
local gene transfer in the mouse has been successfully
employed thus far. Recombinant adenoviruses, injected
intracardially into early stage embryos, uniformly infect
the entire vascular bed. Use of viruses expressing a ßgalactosidase marker gene allows visualization, whereas
use of viruses encoding (anti)-angiogenic genes allow
genetic manipulation of the endothelial or periendothelial
cells in the embryonic vasculature [26,31]. Later during
embryonic development and after birth, systemic injection
of recombinant adenoviruses results in the preferential
infection of the liver parenchyme with only minimal
infection of the systemic endothelium. Cationic liposome–
DNA complexes may be better vehicles for targeting
transgenes to the endothelium in an organ-specific pattern
[81]. Endothelial cells within certain organs [82] or tumors
[83] express specific markers, a property that can be
exploited for targeting (anti)-angiogenic factors.
1.5. Technical considerations
The number, length and surface area of blood vessels,
and their constituent cells (endothelium and pericytes /
smooth-muscle cells) or extracellular matrix can be analyzed by standard histological and immunocytochemical
methods. Endothelial cells in sprouting vessels fail to stain
for von Willebrand factor, but are immunoreactive for
CD31, CD34, lectin, VE-cadherin, thrombomodulin or the
a v ß 3 [37,84–87] integrin. Other endothelial markers include FLK-1 FLT-1, TIE-1 or TIE-2 [88,89], whereas
FLT-4 identifies lymphatic endothelial cells [19]. Smoothmuscle a-actin, desmin, vimentin, smooth muscle myosin,
tropomyosin, or the PDGFR-ß mark pericytes but their
expression pattern varies in different vascular beds and, in
addition, these markers frequently identify vascular smoothmuscle cells [90–94]. Morphological and functional
characteristics of the endothelial cells in vivo (e.g.the size,
shape, border and number of endothelial cells; the number
and distribution of endothelial gaps; the sites and degree of
the permeability; or the attachment of leukocytes) can be
studied using silver nitrate, Monastral blue B, Evans blue,
India ink, radioactively labelled tracers or fluorescent
microspheres [95–101]. The three-dimensional pattern of a
vascular network can be visualized by whole mount
immunostaining or in situ hybridization [16], by injection
of intravascular markers (colloidal carbon, India ink,
radioactively labeled red blood cells or albumin, fluorescent high-molecular weight tracers or liposomes)
[27,36,99], by barium-gelatin angiography [2], by vascular
casting [33,96], or, hopefully in the near future, by
magnetic resonance imaging. Its pattern can be reconstructed via computer-assisted imaging [15,102]. Blood
flow can be monitored noninvasively using Doppler ultrasound [103], laser Doppler [76], microvascular videomicroscopy [12], magnetic resonance imaging [104], or,
invasively, by colored microspheres or radioactive tracers
[105,106]. Shear stress, direction and speed of the blood
flow, capillary perfusion, and cell–cell interactions
(leukocyte rolling, extravasation) can be measured using
intravital videomicroscopy [12]. Interactions between
blood-borne cells (leukocytes, tumor cells) and vascular
endothelium can be studied by prelabeling the circulating
cells with fluorescent dyes [107], or using fluorescent dyes
which selectively attach to neutrophils (acridine orange)
[108]. Endothelial cell proliferation can be evaluated by
quantitative autoradiography of incorporated 3 H-labeled
thymidine, or by histological analysis of proliferation
markers (Ki67, PCNA) or of incorporated 59-bromo-29deoxyuridine (BrdU) [109]. Endothelial cell death can be
analyzed in situ using vital dyes, routine light microscopy
or electron microscopy, the TUNEL method [34], or
biotin-labeled Annexin V [110].
1.6. Transgenic mouse models
Several transgenic mouse models of perturbed vascular
development have been developed over the last years.
Abnormal embryonic vascular development, resulting from
defects in the formation of a primitive capillary plexus, has
been observed in mice lacking VEGF [27], FLT-1 [20],
FLK-1 [22], TGF-ß [111], fibronectin [112], or VEcadherin [113]. Defects in the expansion and remodeling of
the embryonic vasculature occur in mice deficient in TIE-1
[21], TIE-2 [114], angiopoietin-1 [115], Braf [116], HIF1a (unpublished observations), ARNT [117,118], VHL
[119], VCAM-1 [120], the a 4 integrin [121], or in mice
overexpressing neuropilin [122], or angiopoietin-2 [23].
Impaired recruitment and investment of mural cells has
been observed in mice with disruption of the genes
encoding PDGF-B [91], PDGF-B receptor [123], tissue
factor [24], and LKLF [124], whereas reduced formation
of extracellular matrix occurs in mice lacking collagen
type I [125].
Abnormal blood vessel development or function after
birth occurs in mice lacking collagen type III [126] (vessel
fragility with bleeding), P-selectin [54] (reduced leukocyte
rolling), or fibrillin-1 [127] (aneurysmal dilatation and
rupture). Mice overexpressing VEGF in the retina [128], or
mice with sickle cell disease [129] develop retinal and
choroidal neovascularization, whereas overexpression of
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
the Fps / Fes tyrosine kinase [68] or the Polyoma middle-T
antigen [69,70] results in the formation of hemangiomas.
Overexpression of VEGF-C in the skin induces lymphangiomas [130], while overexpression of the bovine polyoma
virus-1 genome [66], the simian virus large T-antigen [67],
or conditional overexpression of VEGF [131] results in the
formation of vascularized tumors.
2. Arterial stenosis and reendothelialization
Arterial injury such as occurs after balloon angioplasty
or placement of intravascular stents frequently results in
chronic narrowing of the lumen due to constrictive remodeling of the vessel wall and intimal accumulation of
smooth-muscle cells and matrix [132,133]. Few long-term
treatments are available, mandating a better understanding
of this process at the genetic level. To date, only a few
mouse models of arterial injury have been developed,
despite significant efforts (Table 1). The mouse differs
from other species by the thinness of its arteries (only 2 to
3 smooth-muscle cell layers and elastic laminae are present
in the media), by the absence of smooth-muscle cells in the
intimal layer, by the lack of side-branches in the carotid
artery, and, last but not least, by its small size (the lumen
of the carotid artery is |300 mm wide).
2.1. Mechanical injury models
Based on a widely used mechanical injury model in the
rat [134], a flexible nylon filament wire was used to
mechanically injure the murine carotid artery [135]. This
results in complete denudation of the endothelium, disruption of the internal elastic lamina and focal injury of the
medial smooth-muscle cells (|25% cell death). Thrombosis
or inflammation are minimal to absent. Within 48 h,
residual medial smooth-muscle cells start to proliferate and
to migrate across the elastic laminae, with the first smoothmuscle cells accumulating in the neointima by day 4 after
injury. Proliferation of smooth-muscle cells in the media is
maximal (|10%) within 5 days after injury, and in the
intima (|60%) within 8 days after injury. Despite these
Table 1
Overview of arterial stenosis used in the mouse
Medial necrosis
Thrombosis
Inflammation
Endothelial
denudation
Neointima
formation
Remodeling
Neoadventitia
Mechanical
injury
Electric
injury
Remodeling
model
Perivascular
cuff
25–50%
6
6
1
100%
11
11
1
60%
6
1
2
ND
2
1
2
1
11
11
11
ND
ND
Dilatation
11
Constriction
1
ND
11
ND: Not determined.
15
significant proliferation rates, the (frequently eccentric)
neointima contains only two to three layers of smoothmuscle cells (|120 cells per cross-section), covering an
area of |0.012 mm 2 within 2 weeks after injury. It is
unknown whether arterial remodeling occurs in this model.
Reendothelialization of the denuded area is complete
within 3 weeks after injury.
This model was subsequently modified by using different mechanical devices to denude the endothelium and to
injure the medial cells [136–139]. In our hands, the use of
a guidewire (|350 mm) induces a significant neointimal
response containing several layers of smooth-muscle cells
(|250 cells per cross-section) within 3 weeks after injury
(Fig. 4a, b). This more vigorous response appears to be, at
least in part, due to a more severe injury (|50% medial
cell death). Initial analysis indicates a significant influence
by the genetic background of the mouse, as C57Bl / 6 mice
appear to be less responsive than mice with a mixed
genetic background of C57Bl / 6 and 129. In aggregate, this
model allows the study of the molecular basis of the
smooth-muscle cell response to endothelial denudation and
medial injury in the presence of only minimal thrombosis
and inflammation. In addition, the type of injury mimics
balloon angioplasty, frequently used in humans.
2.2. Perivascular electric injury
To circumvent the technical challenge of inserting
intravascular devices, a perivascular injury model was
developed whereby an electric current is passed through
the wall of femoral or carotid arteries using an electromicrocoagulator [109]. Electric injury induces a severe
injury, destroying all cells across the vessel wall, including
the smooth-muscle cells in the media, the endothelial cells
in the intima and the fibroblasts in the adventita. This
results in transient platelet- and fibrin-rich thrombosis, and
removal of the necrotic cell and matrix debris by infiltrating leukocytes in the intima, media and adventitia during
the first week after injury. Because of the complete cell
necrosis across the injury, healing of the vascular wounds
initiates from the adjacent uninjured borders. Smoothmuscle cells in the adjacent uninjured borders start to
proliferate and to migrate across the internal elastic lamina
into the intima (Fig. 4c). Subsequently, they migrate
alongside the lumen and within the media, thereby repopulating the necrotic wound. A similar pattern of healing
also occurs in end-to-end microvascular anastomoses
[140], venous or prostethic grafts [141], or laser-induced
thermal injury [142]. Sequential analysis of the topographic pattern of cell accumulation in the wound during the
entire time-course of healing allows quantitatation of
smooth-muscle cell migration. Beyond two weeks after
injury, the intima contains on average |13 layers of aactin immunoreactive smooth-muscle cells (|200 cells per
cross-section), covering an intimal area of |0.03 mm 2 and
stenosing the lumen for |30%. Vascular remodeling (e.g.
16
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
Fig. 4. Arterial stenosis models in the mouse. (a) Histological section through a normal femoral artery. (b–d) Within 2 to 3 weeks after injury, a neointima
develops in the femoral artery after mechanical injury (b), perivascular injury (c) or in the carotid artery after perivascular cuff application (d). (e, f)
Neointima formation in a carotid artery transplant, stained with hematoxylin–eosin (e) or smooth muscle a-actin (f).
the adaptive outward displacement of the vessel wall in
response to intimal thickening so that the vascular lumen is
preserved) prevents progressive stenosis of the lumen,
despite increasing neointima formation. A marked neoadventitial response occurs, characterized by accumulation of
leukocytes and fibroblasts, and secondary matrix deposition. Reendothelialization (resulting from both proliferation
and migration of endothelial cells) in this model is rapid
and complete within two weeks after injury. This model
has allowed us to evaluate the specific role of the different
components of the plasminogen system [136,137,143,144].
Notably, qualitatively similar genotypic differences were
obtained when the mechanical injury model was used. In
addition, this model has been used successfully to test the
suppressive effect of adenoviral gene transfer of PAI-1 (the
primary inhibitor of the plasminogen activators) on neointima formation [137]. When the replication-defective PAI-1
adenovirus was injected intravenously, infected parenchymatous liver cells produced large amounts of PAI-1,
resulting in |1000-fold increased plasma PAI-1 levels.
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
Secondarily, PAI-1 was deposited into the developing
neointima, preventing smooth-muscle cell migration and
the formation of a neointima in situ. The wounded artery
was not directly transduced by the PAI-1 adenovirus. Thus,
this model offers the opportunity to study the molecular
mechanisms of smooth-muscle cell migration during conditions of thrombosis, inflammation and matrix remodeling, that are more severe than in the mechanical injury
model (see above) or in the other injury models (ligation
model, perivascular collar, etc; see below). It should be
noticed that fibrin-rich clots, matrix deposits and
leukocytes are frequently present in atherosclerotic arteries
subjected to vascular interventions of angioplasty or
stenting [133,145]. In addition, this model allows the study
of the molecular basis of the physiological (outward)
remodeling in response to intimal thickening.
2.3. Arterial remodeling models
Vascular remodeling is an adaptive process that occurs
in response to chronic changes in hemodynamic conditions
[146]. It involves changes in cell growth, cell death, cell
migration and in extracellular matrix composition, that
lead to a compensatory adjustment in vessel diameter and
lumen area. The blood vessel essentially remodels such
that the lumen area is modified to maintain shear stress (a
reduction in blood flow increases intimal lesion formation
in vascular grafts and balloon-injured vessels) and / or wall
stress (determined by the diameter of the vessel and blood
pressure, and inversely by the vessel wall thickness).
Constrictive remodeling during restenosis may result in
late luminal area loss [147]. Remodeling during atherosclerosis initially involves outward displacement of the
vessel wall so that the vascular lumen is relatively preserved, but once this adaptive mechanism of remodeling is
exhausted, luminal narrowing occurs during advanced
atherosclerosis [148].
To study the molecular mechanisms of arterial remodeling during arterial stenosis, a mouse model was developed,
in which the blood flow (but not the pressure pulsations) in
the common carotid artery is disrupted by ligating the
vessel near the distal bifurcation [149]. The endothelium in
the ligated vessels is not denuded, but becomes detached
from the underlying internal elastic lamina, thereby forming spaces that are filled with red blood cells. Within two
days after ligation, a marked loss of smooth-muscle cells in
the media is evident (|60% cell death). Luminal narrowing
(|80% reduction) occurs in part by formation of a concentric neointima, containing on average |100 a-actin
immunoreactive smooth-muscle cells per cross-section and
covering an intimal area of |0.035 mm 2 . In addition, the
vessel becomes constricted as evidenced by the |25%
reduction in the outer circumference. Thrombosis only
occurs within the first 2 mm of the ligature. Leukocytes
and platelets penetrate through discontinuities in the
endothelial sheet into the subendothelial space, and are
17
detected during the initial postligation period in all layers
across the vessel wall. Possibly, the altered flow conditions
influence the expression of endothelial-leukocyte adhesion
molecules and chemokines, with a subsequent influx of
inflammatory cells. The latter presumably play an essential
role in this model, as ligated P-selectin-deficient arteries
are not infiltrated by leukocytes, and only develop a
minimal neointima without luminal narrowing [149]. This
model thus offers the opportunity to study the molecular
mechanisms (in particular the role of leukocyte–platelet–
smooth-muscle cell interactions) contributing to the formation of intimal lesions in the absence of noticeable
endothelial denudation. In addition, it allows us to unravel
the molecular basis of the vascular constriction, accompanying conditions of perturbed flow and turbulence.
Vascular remodeling during atherosclerosis was examined in mice lacking the apolipoprotein E (Apo-E) alone
[107], or in combination with the low-density lipoprotein
(LDL) receptor [150]. Despite significant intimal thickening, the lumen in the atherosclerotic aorta was not smaller
because of outward displacement of its vessel wall.
However, in the femoral and carotid arteries, the lumen is
constricted, possibly related to the media necrosis and
adventitial inflammation [107]. Endothelium-dependent
relaxation to receptor- and nonreceptor-mediated agonists
was significantly impaired in the atherosclerotic vessels
[150]. We have also observed that the vascular lumen in
the aorta from mice lacking ApoE, ApoE and the plasminogen activator (PA), tissue-type PA (t-PA) or
urokinase-type PA (u-PA) remains relatively preserved
because of adaptive remodeling, despite large intimal
plaques [151]. However, vascular remodeling in the ApoEand ApoE:t-PA-deficient but not in the ApoE:u-PA-deficient aorta was accompanied by additional destruction of
the media, resulting in aneurysm formation (see below)
[151]. Why media necrosis results in luminal stenosis in
peripheral arteries, in contrast to the aneurysmal dilatation
in the aorta, remains to be further determined.
2.4. Other arterial stenosis models
Other models have also been used to induce intimal
hyperplasia, but have not been characterized in detail or
are still under investigation. A perivascular cuff model
based on the use of a nonconstricting hollow polyethylene
tube (similar to that used in the rabbit [152]), induces a
significant neointima in mice within three weeks [153]. In
this model, the endothelium is not denuded, there is only
minimal thrombosis and smooth-muscle cell death, and the
elastic laminae remain structurally intact. A concentric
neointima, consisting of 8 to 10 smooth-muscle cell layers
and a significant neoadventitia, characterized by active
neovascularization, develop within 3 weeks (Fig. 4d).
Although the precise mechanisms responsible for intimal
thickening in this mouse model remain undetermined,
previous studies in other species have suggested that this
18
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
may result from endothelial activation, kinking of the
artery, vasospasms, increased flow velocity, blockage of
the lymphatic drainage, damage of the adventitial nerves of
vasa vasorum (resulting in hypoxia of the media), or
chemical toxicity of the collar [152]. Leukocytes presumably play a central role in triggering the smoothmuscle cell response.
Another model involves the surgical removal of an
intimal / medial fragment of the carotid artery using an
intraluminal tungsten wire to induce an intimal response
(P. Yiu and J. McEwan, personal communication). Smoothmuscle cells migrate through the breaks in the elastic
laminae into the intima to form an eccentric intimal lesion,
which is proportional to the depth of the injury. In a
venous graft model, a fragment of an autologous vein is
surgically sutured into an arteriotomy in the carotid artery
(V. Shi, personal communication). Activation or dysfunction of the grafted venous endothelium by the arterial
blood pressure may play a role in the induction of the
smooth-muscle cell response and intimal thickening of the
graft. Although reproducible, this model is technically
challenging and the lack of clearly visible elastic laminae
in the venous graft may hamper morphometric quantitation. Other models relying on the use of external crush, air
desiccation or cryoinjury have been attempted without
much success. Photochemical dyes (such as Rose Bengal)
induce controllable endothelial denudation and medial
injury, once they are activated by transluminal green light,
and have been used to induce an intimal response in other
species [154]. Their usefulness in mice is currently being
tested.
2.5. Gene transfer in murine arteries
Local gene transfer in the vessel wall of larger species
has been based on the use of recombinant adenovirus
[155], adeno-associated virus [156], liposomes [157,158],
naked DNA [159,160], or on the seeding of retrovirallytransduced endothelial [161] or smooth-muscle cells [162].
Due to its small size, local gene transfer in the murine
arteries has not been achieved yet, and systemic injection
of adenovirus does not result in significant transduction of
the quiescent or injured murine artery [137]. However,
systemic gene transfer may be successfully used in the
mouse. Indeed, when recombinant adenovirus is systemically injected in the mouse, it preferentially transduces
parenchymatous liver cells which subsequently produce
large amounts of the transgene. This results in plasma
levels of the expressed protein of 10 to 100 mg per ml for
at least 7 to 10 days [137]. An advantage of such systemic
gene transfer is the absence of local inflammation or
perturbed gene expression in the artery, as occurs after
local adenovirus-mediated gene transfer in larger species
[163]. The feasibility of such a strategy was demonstrated
by the inhibitory effect of adenovirus-mediated gene
transfer of PAI-1 [137] or ApoA-I [139] on neointima
formation in mice. Use of the second generation adenovirus vectors, which prolongs expression of the transgene
up to several months, may even further improve the
applicability of this strategy [164].
2.6. Transgenic mouse models
Mice with genetic deficiencies of the different components of the plasminogen or matrix-metalloproteinase
system have been used to analyze the wound-healing
response of mechanically or electrically injured arteries
[136,137,143,144,165]. Notably, both injury models revealed a similar requirement for urokinase-generated plasmin proteolysis in migration of smooth-muscle cells and
tissue remodeling by infiltrating leukocytes. Furthermore,
systemic overexpression of their inhibitor PAI-1 by intravenous injection of a recombinant adenovirus expressing
PAI-1, reduced arterial neointima formation [137]. The
response to mechanical injury has also been studied in
mice lacking the estrogen receptor-a [138,166] or
apoliprotein E [139]. In the latter study, a significant
suppression of neointima formation by adenovirus-mediated transfer of the apolipoprotein A-I gene was reported
[139]. The perivascular cuff model has been used to
characterize the intimal response in mice lacking endothelial nitric oxide synthase [153]. The ligature model was
used in P-selectin-deficient mice [149].
Taken together, the transgenic mouse offers a unique
opportunity to unravel the molecular basis of the intimal
response at the genetic level. Despite significant progress,
the currently available mouse models suffer shortcomings
and require further optimization. (i) Uninvolved arteries
have thus far been selected for injury, and it is likely that
the arterial wound-healing response in diseased (atherosclerotic) arteries, such as in the atherosclerotic apoE- or
LDLr-deficient mice, will differ significantly. (ii) Arterial
stenosis in patients does not only result from intimal
thickening but also from arterial wall remodeling (except
during stent stenosis, when shrinkage of the vessel wall is
prevented). Arterial remodeling was only characterized in
the perivascular electric injury model (in which it results in
lumen expansion instead of narrowing) and in the ligation
model (in which remodeling induces constriction of the
lumen). No information is available on the type or degree
of remodeling in the other models. (iii) Local (gene-)
transfer in the mouse artery remains a technical challenge.
(iv) None of these models, which differ significantly in
their mechanisms, ideally mimic the human disease process. A more in-depth analysis and comparison between
the various injury models will increase our understanding
of the role of candidate disease molecules in the various
aspects (cellular migration, proliferation, matrix remodeling etc.) of the arterial-healing process. Indeed, the mechanical injury models are suited to the study of the
intimal response in the absence of thrombosis and inflammation, whereas the perivascular electric injury model
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
allows the study of intimal thickening induced by a more
severe injury associated with thrombosis and inflammation.
The ligation and collar models, on the other hand, allow
the study of the role of platelet–leukocyte interactions in
the intimal response in the absence of endothelial denudation.
3. Transplant-induced arteriosclerosis
Transplant-associated arteriosclerosis is the major cause
of cardiac allograft failure after the first postoperative year,
and it appears to be a significant problem in the long-term
survival of other solid organ transplants [167]. In contrast
to the hypercholesterolemia-induced atherosclerosis, the
lesion associated with transplant arteriosclerosis involves
the artery in a concentric rather than an eccentric fashion,
lipid accumulation is less common in its early development, and progression of the disease occurs at a faster
tempo. It has been proposed that the parenchymal rejection
and vasculopathy are caused by the recipient’s immune
response to foreign antigens, presented on the cells of the
allograft.
3.1. Carotid artery transplant model
A murine model of allograft transplantation has been
developed in which carotid arteries are transplanted between a B.10A(2R) (H-2 h2 ) donor mouse and a C57BL / 6
(H-2 b ) recipient mouse, without the use of immunosuppressive drugs [168]. Grafts are paratopically transplanted in an end-to-side anastomosis in two longitudinal
arteriotomies, made in the recipient’s carotid artery. Only
grafts with prominent pulsations (indicative of patent flow
and lack of thrombosis) are studied. The endothelium
remains structurally intact and apposed to the intima
throughout the entire period after transplantation. Within 7
days, the allografted carotid artery forms a three- to sixcell-thick neointima composed of recipient-derived
leukocytes (predominantly monocytes or macrophages, and
to a lesser extent, CD4 1 helper and CD8 1 cytotoxic
lymphocytes). At 15 days, a third of the neointimal cells
are CD45 immunoreactive leukocytes, but by 30 days, the
concentric neointima contains predominantly smooth-muscle cells (|800 cells per cross-section) (Fig. 4e, f). The
neointimal accumulation of smooth-muscle cells occurs
coincidently with their replacement in the media by
nuclear debris and leukocytes which infiltrate through
breaks in the elastic laminae. This sequence of events
suggests that leukocytes degrade the elastic laminae and
infiltrate in the media, activate the medial smooth-muscle
cells (presumably by producing cytokines, chemotactic
agents and growth factors such as for example PDGF-B
and FGF-2), and induce their migration into the intima.
Plasmin proteolysis appears essential, as the elastic
laminae fail to become degraded, leukocytes are not
19
infiltrating into the media, and neointimal smooth-muscle
cell accumulation is significantly impaired in wild-type
carotid arteries, transplanted into plasminogen-deficient
recipients (unpublished observations).
Subsequent studies in mice deficient in RAG-2 (which
lack an antigen-specific cellular and humoral immune
response), in mMT (in which B cells are depleted and the
humoral immune response is deficient), in CD4 (lacking
CD4 1 T cells), in MHC class II (depleted of CD4 1 T
cells), in MHC class I (depleted of CD8 1 T cells), or in the
mutant beige mice (bg /bg; in which the natural killer cells
are depleted) or osteopetrosis mice (op /op; lacking macrophage colony-stimulating factor and depleted of macrophages) revealed that transplant arteriosclerosis depends on
T-cell receptor and immunoglobulin gene rearrangement
(both consequences of active immunization), but not on
CD8 1 T cells and natural killer cells [169]. Development
of the neointima requires the interaction of CD4 1 T cells,
B cells and macrophages, as a reduction in the number of
mature CD4 1 T cells and macrophages, and of the
probable absence of allospecific antibodies results in the
inhibition of smooth-muscle cell proliferation and / or
migration. In another study, luminal occlusion and crosssectional neointimal area were found to be greater in
arteries allografted into hypercholesterolemic ApoE-deficient recipients at 15 and 30 days after transplantation.
This appears to be attributable to an increased accumulation of smooth-muscle cells and, to a much lesser extent,
of leukocytes, collagen or lipid in the intima [170].
Possibly, the greater smooth-muscle cell response results
from an increased production of growth factors by macrophage-derived foam cells than by nonlipid-containing
macrophages. Appreciable outward remodeling of the
arteries, allografted in the ApoE-deficient recipients, occurs, presumably in response to the excessive neointima
formation.
A model of chronic rejection using aortic transplantation
from C3H, B10.BR or C3H.SW donors into C57BL / 10
recipients has been developed [171]. Mice, free from
complications 12-h postoperatively, had long-term survival
and developed a marked intimal thickening, rich in smoothmuscle cells, in the aorta transplant after 2 months [171].
3.2. Cardiac transplant model
In order to study the myocardial rejection in addition to
the graft coronary vasculopathy, a model has been developed in which mouse hearts are transplanted heterotopically from B10.1 to B.10.BR mice (class I MHC antigen
disparity), from bm12 to C57BL / 6 mice (class II MHC
antigen disparity), or from 129 to C57BL / 6 (nonMHC
antigen disparity) [172]. This involves ligation of the
inferior and superior vena cavae and the pulmonary veins
of the donor heart, and anastomosing the donor aorta and
pulmonary artery to the recipient abdominal aorta and vena
cava, respectively. Transient immunosuppression with anti-
20
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
CD4 and -CD8 antibodies permits graft survival. Donor /
recipient antigenic differences may be either class I or
class II major histocompatibility antigens (H-2) or nonH-2
antigens. Initially, an infiltrate of CD4 1 and CD8 1 T
lymphocytes and macrophages of recipient origin concentrates in the intima and adventitia of larger coronary
arteries, with little involvement in the myocardium. Subsequently, the intima expands with (primarily) smoothmuscle cells of donor origin, and the media becomes
infiltrated by leukocytes. It appears that T cells and
heterogeneous antigens in the endothelium and / or the
media play an essential role in the induction of graft
arteriosclerosis in this model. Transplants between strains
that produce antibodies to donor cells (B10.A to B10.BR),
develop coronary lesions exceeding those in the reverse
combination, in which no detectable antibodies are detected, even though their histoincompatibility is similar
[173]. This suggests that humoral immunity is also implicated in the graft arterial disease. Transplantation of 129
allografts into ApoE-deficient C57BL / 6 recipients results
in an accelerated graft arteriosclerosis, revealing a significant proatherogenic effect of the hyperlipidemic environment [174]. This model has been used to determine the
suppressive effects of IFN-g- [175], ICAM-1- or LFA-1specific antibodies [176] on transplant arteriosclerosis. A
stimulatory effect of IFN-g was also observed in another
study, in which hearts from C-H-2 bm12 KhEg (H-2 bm12 )
donor mice were transplanted into C57BL / 6 (H-2 b ) IFNg-deficient recipients after immunosuppression with antiCD4 and anti-CD8 antibodies [177]. In wild-type recipients, myocardial rejection peaked at 4 weeks, and by 8 to
12 weeks, coronary arteriopathy evolved. In contrast, graft
arterial disease did not develop in IFN-g-deficient recipients, despite a marked myocardial rejection, indicating that
development of graft arterial disease, but not parenchymal
rejection, requires IFN-g.
In another model, hearts are heterotopically transplanted
between DBA / 2 (H-2 d ) donor mice to B10.D2 (H-2 d )
recipient mice, which share major histocompatibility antigens but differ in their minor antigens [178]. Significant
survival of allografts (70%) was achieved without immunosuppression. Concentric intimal hyperplasia, comprising |40% of the graft arterial wall, together with interstitial and perivascular fibrosis were present. The endothelium appeared intact. The grafts that failed to survive,
exhibited features of interstitial edema, haemorrhage,
myocardial necrosis and severe mononuclear cell infiltration with diffuse fibrosis, indicative of acute rejection.
There is no apparent correlation between the severity of
rejection and the degree of vessel disease.
4. Hypercholesterolemia-induced atherosclerosis
Atherogenesis is a complex process in which the lumen
of a blood vessel becomes narrowed by cellular and
extracellular substances [179]. Lesions progress mainly
through three stages. The first stage is the fatty streak
lesion, which is characterized by the presence of lipidfilled macrophages (foam cells) in the subendothelial
space. The second stage is the fibrous plaque, which
consists of a central acellular area of lipid, derived from
necrotic foam cells, covered by a fibrous cap containing
smooth-muscle cells and collagen. The final stage is the
complex lesion, in which plaque rupture triggers the
clinical event of thrombus formation with deposition of
fibrin and platelets, and in which media destruction may
lead to aneurysm formation and fatal bleeding after
rupture.
The wild-type laboratory mouse is highly resistant to the
development of atherosclerotic plaques, presumably related
to a different distribution of cholesterol among its lipoproteins [180]. Other mouse-specific differences include absence of the cholesteryl ester transfer protein (CETP) and
of lipoprotein (a), and different editing of apolipoprotein
(Apo)B-100 [180]. Initial studies revealed that significant
strain-dependent differences determined the susceptibility
to atherosclerosis [180]. The first available mouse models
of atherosclerosis, including the C57BL / 6 mice fed a
cholesterol- and cholate-rich diet, suffered significant
shortcomings. Indeed, even after prolonged feeding on an
unphysiological diet, containing 10 times the cholesterol of
a Western-type diet and the unnatural dietary constituent
cholic acid (which regulates removal of cholesterol from
the body), these mice only developed atypical and immature early fatty streak lesions with a restricted distribution,
casting doubt on their overall relevance to human disease.
Furthermore, as this diet caused a chronic inflammatory
state, it might have induced perturbation of the interplay of
immune cells and cytokines involved in atherogenesis.
With the advent of transgenic mice, over- or under-expressing other candidate transgenes, and the further manipulation of gene expression via bone marrow transplantation or adenovirus-mediated gene transfer, significant
progress has been achieved in unraveling the molecular
mechanisms of this disease process (Fig. 5).
4.1. Lipid metabolism
The most widely studied mouse model of atherosclerosis
is a transgenic mouse strain, deficient in apolipoprotein E
(ApoE) [181,182]. This molecule is a ligand that mediates
low-density lipoprotein (LDL) receptor clearance of
chylomicrons, very-low-density lipoproteins (VLDLs) and
other serum lipoproteins. Consequently, ApoE-deficient
mice spontaneously develop hyperlipidemia (predominantly VLDL) and elevated cholesterol levels (|500 mg / dl) on
a low cholesterol / low fat diet. They develop lesions of all
phases with morphological features closely resembling
human lesions at vascular sites typically affected in human
atherosclerosis, e.g. at the base of the aorta, the lesser
curvature of the thoracic aorta, at several branch points of
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
21
Fig. 5. Overview of the different atherosclerosis models in the mouse. The most widely used models (apoE- or LDLr-deficient mice) can be crossbred into
other transgenic mouse strains with over- or under-expression of other atherosclerosis modifier genes.
the carotid, intercostal, mesenteric, renal and iliac arteries,
and in the proximal coronary, carotid, femoral, subclavian
and brachiocephalic arteries [183,184]. Adherent monocytes are present on the surface and migrate across the
endothelium before the development of foam cell-rich fatty
streaks. As the lesions progress, smooth-muscle cells form
a fibrous cap rich in collagen and elastin fibers over the
foam cell-rich areas, and cholesterol clefts, calcifications
and necrotic areas appear within the core regions of the
fibrous plaques. Lumen narrowing and occlusion with
associated myocardial fibrosis (indicative of ischemia) are
observed in the most advanced stages. Notably, this
phenotype does not depend on complete absence of ApoE,
as heterozygous ApoE-deficient mice also develop fibrous
lesions on a high-fat / high-cholesterol diet [185,186].
Cell surface LDL receptors (LDLr) play a fundamental
role in regulating plasma cholesterol levels by mediating
cellular uptake of LDL and intermediate-density lipoproteins (IDLs). Defects in the LDLr gene are the best
documented genetic causes of premature atherosclerosis in
humans. LDLr-deficient mice on a normal chow diet have
only mild pathological lesions, with slightly elevated
plasma cholesterol (IDL / LDL fraction), a phenomenon
attributed to the presence of an alternative ApoB-II pathway for LDL clearance in the mouse [187,188]. However,
when fed an atherogenic diet, these mice develop significant fatty streak lesions containing a lipid-filled necrotic
core. Fibrous lesions are, however, absent. Combined
ApoE:LDLr-deficient mice suffer more severe hyperlipidemia and lesion formation [150].
Several additional transgenic strains have been generated that over- or under-express modifier genes, able to
modulate the lipoprotein metabolism and the development
of atherosclerotic lesions (Table 1). These include transgenic mice overexpressing ApoE Leiden [189,190],
ApoE R142C [191,192], ApoA-II [193], CETP [194], lecithin
cholesterol acyltransferase (LCAT) [195], human apolipoprotein (a) [196], or human ApoB-100 together with the
apolipoprotein (a), which results in the formation of
lipoprotein (a) or Lp (a) [197]. Other modifiers have been
evaluated by overexpression on a wild-type or on a ApoEor LDLr-deficient background, including ApoA-I (ApoEdeficient background [198–200]), ApoA-IV (wild-type
background [201]; apoE-deficient background [202]), or
Apo C-III (wild type background [203]; LDLr-deficient
background [204]). Additional transgenic mice, generated
via gene targeting, lack ApoA-I [205], LCAT [206],
ApoC-I [207], hepatic lipase [208], the macrophage type I
and type-II class-A scavenger receptor [209], or Apobec-1
[210,211]. Other targeted mice exclusively express
ApoB48 or ApoB100 [212], or a human ApoE3 isoform
instead of the normal ApoE allele [213].
Bone marrow transplantation studies offer the opportunity to further manipulate the lipid metabolism. Indeed,
bone marrow from a wild-type donor has been shown to
correct the hyperlipidemia and to prevent atherosclerosis in
ApoE-deficient hosts [214–216]. In addition, local production of ApoE by expression of a transgene in macrophages or in the blood vessel wall diminishes atherosclerosis, independent of its cholesterol-lowering effect,
22
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
revealing the importance of local ApoE production [217].
Use of recombinant adenoviruses expressing ApoE results
in normalization of the lipid and lipoprotein profile with
markedly decreased total cholesterol, VLDL, IDL and
LDL, and increased HDL [218]. Adenoviral gene transfer
of ApoE isoforms in ApoE-deficient hosts reveals a
significantly impaired ability of ApoE2 to mediate clearance of remnant lipoproteins as compared to ApoE3 or
ApoE4 [219]. Notably, use of second generation adenovirus expressing ApoA-I results in significantly higher
levels of ApoA-I for a longer time in LDLr- than in
ApoE-deficient mice [220].
4.2. Proteinases and extracellular matrix
Stable atherosclerotic lesions can develop insidiously for
prolonged periods without medical threat. However, when
plaques become unstable, they may rupture and trigger the
onset of myocardial ischemia because of occluding thrombus formation [145]. Unfortunately, the currently available
models of atherosclerosis in the mouse (and in other
species) fail to progress to spontaneous plaque rupture. In
the mouse, this failure may be attributable (at least in part)
to the lower arterial wall stress, and / or to the absence of
other risk factors (hypertension, smoking, diabetes etc).
Nevertheless, the mouse may be a valuable model to study
some of the mechanisms contributing to plaque instability,
as evidenced by the observation that arterial calcification is
under genetic control [221,222]. Widespread calcified
cartilaginous metaplasia occurs within spontaneous
atherosclerotic lesions in ApoE-deficient mice [222].
Spontaneous calcification of all elastic and muscular
arteries (but not the arterioles, capillaries or veins) was
also observed in mice lacking the matrix-GLA protein
(Mgp), a protein synthesized by vascular smooth-muscle
cells [223]. Mgp-deficient mice develop till term but die
within two months as a result of rupture of the thoracic and
abdominal aorta. Noticeably, arterial calcification occurs in
the absence of atherosclerotic plaque formation, indicating
that lipid accumulation and calcification are regulated by
independent genetic mechanisms. Another modifier of
calcification is the macrophage colony-stimulating factor
(M-CSF), as evidenced by the finding that M-CSF:ApoEdeficient mice develop significant arterial calcification,
even in the absence of advanced plaques [224]. Urokinasetype plasminogen activator (u-PA) may also be implicated,
as ApoE:u-PA-deficient mice develop plaques with an
increased number of microcalcifications (unpublished observations).
Little information exists on the amount or nature of the
extracellular matrix in atherosclerotic plaques in the various available transgenic mouse models. Mice deficient in
ApoE and u-PA on a high-fat / high-cholesterol / cholate diet
have similar plaque growth as ApoE-deficient mice, but an
increased deposition of collagen-rich matrix at the ultrastructural level (unpublished observations). Accelerated
plaque progression has been observed in mice with a
combined deficiency of ApoE and plasminogen (Plg)
[225]. Although it was anticipated that deficiency of Plg
would result in an increased deposition of matrix, similar
amounts of fibrin were present in ApoE- and in ApoE:Plgdeficient mice. The precise mechanism of the increased
plaque growth remains thus unclear. A particular problem
with this study is, however, that deficiency of Plg lowers
the HDL levels by 75%. Whether this relates to the poor
general health or altered immune / inflammatory status of
these mice remains to be determined. As HDL is an
important determinant for plaque growth in mice, the
accelerated growth in the mutant mice may have resulted
from an indirect effect on plasma lipoproteins rather than a
direct local effect within the plaques.
Another life-threatening complication of atherosclerosis
is the development of media destruction and aneurysm
formation with resultant fatal bleeding. Although a major
health threat in the elderly, its pathogenetic mechanisms
remain poorly understood [226,227]. Media destruction has
been occasionally detected in advanced atherosclerotic
lesions in LDLr- or ApoE-deficient mice on a high-fat /
high-cholesterol diet [183]. The incidence and severity of
these destructive lesions, and their progression to rupturing
aneurysms appears to be increased by cholate supplements
to the diet [151]. Indeed, media destruction frequently
occurs in lesions beyond 15 weeks of age, progressing in
|10% of the cases to aneurysmal dilatation and pseudoaneurysm formation [151] (Fig. 6). Despite rupture of the
entire aortic wall, bleeding is prevented by compensatory
formation of a fibrous cap in the adventitia. Aneurysmal
dilatation is more severe and frequent at the infra-renal
level in the abdominal aorta than in the thoracic aorta,
similar to the human pathology. Proteinases, produced by
macrophages, play an important role in this process as loss
of urokinase gene function in ApoE-deficient mice prevents media destruction and aneurysm formation, likely via
reduced activation of matrix metalloproteinases [151].
4.3. Immune modulation
It is becoming increasingly evident that immune mechanisms influence atherosclerosis [228]. Human atherosclerotic lesions consistently contain macrophages and T
lymphocytes, two cell types that interact with each other to
promote cell-mediated immune responses. Macrophages
may present antigen to the T lymphocyte within the vessel
wall, whereas T-lymphocyte-derived cytokines may activate macrophages. In addition, antibody responses to heatshock proteins or to modified lipids found in atheromas are
characteristic of the disease. It has been proposed that T
cells recognize peptides derived from oxidatively modified
low-density lipoprotein (oxLDL), thereby promoting B-cell
activation and production of anti-oxLDL antibodies.
Studies in C57BL / 6 mice with antibody immunodepletion of CD4 T lymphocytes [229], and various genetic
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
immunodeficiencies, including SCID mice, athymic nude
mice and mice with class I or II MHC deficiencies [230],
all developed typical fatty streak lesions when fed diets
high in saturated fats, cholesterol and cholate. An increase
in lesion area was observed in the class I MHC-deficient
mice as compared to normal mice. However, the use of
cholate and the absence of T-lymphocytes in the small
lesions in the aortic root of C57BL / 6 mice confound the
interpretation of these studies with regard to the impact of
the immune system on atherogenesis.
Recombinase activator gene (RAG)-2 deficiency results
in a total deficiency of B and T lymphocytes. RAG2:ApoE-deficient mice have no serum autoantibodies, no T
lymphocytes, and a markedly reduced number of MHC
class II-positive macrophages in their atherosclerotic lesions (T lymphocyte-derived IFN-g and IL-4 increase the
expression of MHC class II on macrophages) [231].
Despite these differences, atherosclerotic plaque size is
comparable to that in immunocompetent littermates, indicating that the absence of autoantibodies and T lymphocytes does not influence the extent of aortic lesions in
ApoE-deficient mice. Similar results have been reported in
RAG-1:ApoE-deficient mice with immature, dysfunctional
B and T lymphocytes [232].
The role of IFN-g in atherosclerosis has remained
controversial. On the one hand, it stimulates the expression
of VCAM-1 on endothelial cells, MHC-II on macrophages
and smooth-muscle cells and lipoprotein receptors in
smooth-muscle cells, all potentially proatherogenic properties. On the other hand, it decreases lipoprotein receptor
expression on macrophages, decreases collagen synthesis
in smooth-muscle cells and blocks smooth muscle proliferation, all potentially antiatherogenic effects. Mice with a
combined deficiency of IFN-g and ApoE exhibit a substantial reduction in atherosclerotic lesion size and a 60%
reduction in lipid accumulation, presumably resulting from
an increase of atheroprotective phospholipid /ApoA-IV-rich
particles [233]. In addition, the cellularity of the atherosclerotic lesions is significantly reduced, with a concomittant increase in extracellular collagen content. Whether
these changes result from alterations in smooth-muscle-cell
collagen synthesis, collagenase activity, or cell survival
remains to be determined. Whatever the mechanisms, IFNg promotes atherosclerosis through both local effects in the
vessel wall as well as via a systemic effect on plasma
lipoproteins.
Macrophage colony-stimulating factor (M-CSF) regulates the differentiation, proliferation and survival of
mononuclear phagocytes, functions as a chemotactic agent
for monocytes, and influences the effector functions of
mature monocytes and macrophages. Its expression is
induced in the atherosclerotic wall. Crossbreeding of
ApoE-deficient mice with a mouse line carrying a spontaneously mutated M-CSF gene (op) (resulting in fewer
monocytes and tissue macrophages) yields mice with
higher cholesterol levels than the ApoE-deficient line, yet
23
with significantly smaller and more immature, but more
calcified, atherosclerotic lesions in the aorta [224,234].
Although these data indicate an important proatherogenic
effect of macrophages, it remains unsolved whether the
effects of the op mutation result from decreased circulation
monocytes, reduced tissue macrophages, or diminished
arterial M-CSF.
Tumor necrosis factor (TNF) is a monocyte / macrophage-derived cytokine, able to alter lipid metabolism by
decreasing the activity of adipocyte-derived lipoprotein
lipase and by increasing the production of hepatic VLDL in
response to endotoxin, both being potentially
proatherogenic effects. However, TNF induces nitric-oxide
synthase production and inhibits lipoprotein lipase in the
arterial wall, both potentially anti-atherogenic effects.
C57BL / 6 mice lacking the tumor necrosis factor receptor
p55, develop aortic sinus fatty streak lesions, which are
three-fold larger than in wild-type mice, despite comparable plasma lipid levels [235]. The accelerated atherosclerosis in p55 null mice appears attributable to increased
expression of the scavenger receptor with resultant increased uptake and degradation of acetylated LDL.
4.4. Glucose metabolism
Individuals with diabetes are at increased risk for
developing cardiovascular disease, but the pathogenetic
mechanisms remain poorly understood. Genetically obese
mice with differing severity of obesity, hyperglycemia,
hyperinsulinemia, insulin resistance and islet hyperplasia
and atrophy (fat, obese, tubby, diabetes, and lethal yellow
strains) developed similar or reduced fatty streak lesion
formation on a high-fat / high-cholesterol diet [236]. Notably, protection against accelerated atherosclerosis in
these genetically obese mice appears to result from a
concomittant increase in anti-atherogenic plasma HDL-C
levels. This is the case, regardless whether these mice were
congenic on a C57BL / 6 or on the more diabetogenic
C57BL / Ks background. The importance of HDL in determining lesion development in the mouse is further
illustrated by chronic alcohol feeding of C57BL / 6 mice,
which markedly inhibits the development of fatty streak
lesions, concomittant with a reduction in plasma HDL
cholesterol [237].
More recently, a murine model of accelerated atheroslerosis in diabetic LDLr-deficient mice has been developed. LDLr-deficient mice, rendered hyperglycemic
(glucose.300 mg / dl) by streptozocin treatment and fed a
regular chow diet, have similar cholesterol and triglyceride
levels as normal mice [238]. In hyperglycemic mice,
accelerated formation of irreversible Advanced Glycation
Endproducts (AGEs) occurs, with concomittant induction
of their cellular receptor (RAGE) in the aortic wall. At 6
weeks of chow diet, lesion area is 4-fold increased in
diabetic versus normal LDLr-deficient mice, a process that
can be suppressed by treatment of these mice with
24
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
recombinant soluble RAGE (a competitive antagonist of
RAGE) [239].
4.5. Technical considerations
Several methods have been developed to quantitate
lesion development in the murine aorta. The extent of
atherosclerosis in murine models has usually been determined from cross-sections at a single predilection site
(the aortic root), using modifications of a technique,
originally described by Paigen [240]. This method uses
sequential sections through the heart and aortic origin, and
determines the lesion area in a defined number of sections,
beginning from an anatomical reference point such as the
aortic valve leaflets. An improved variation of this method
using computer-assisted evaluation of 60 progressive sections covering the initial 1.2 mm of the aortic region has
been reported [241]. An advantage of this model is that the
lesion volume can be calculated from the lesion areas,
providing the basis for three-dimensional reconstruction of
the lesions. However, a disadvantage is the atypical site of
atherosclerosis at the aortic root, influenced by the hemodynamic conditions which are different from those in the
thoracic or abdominal aorta. This model is useful in
evaluating differences in atherogenicity in those strains or
under experimental conditions in which lesion formation is
predominantly found in the aortic root.
Another morphometric method quantitates the extent of
atherosclerotic lesions in the entire murine aorta. This
method utilizes computer-assisted image analysis of color
images of Sudan-stained flat (‘en face’) preparations of the
aortic tree (from the heart to the iliac bifurcation), and
determines the percentage of surface area affected by
atherosclerosis [242,243]. The method is rapid and relatively accurate with limited operator dependence. When
compared to the cross-sectional analysis, this method
provides additional information on the shape and the
distribution of these lesions, and, when complemented with
analysis in longitudinal sections [244], on lesion areas and
volumes (at least through the thoracic and abdominal
aorta). This method may be preferred for intervention
studies on large numbers of animals, or in murine models
that develop significant aortic atherosclerosis. Alternative
methods to determine the extent of atherosclerosis in large
groups of mice include the measurement of the unesterified
and esterified cholesterol content in aortic tissue extracts
[245], or the use of 125 I-labeled antibodies specific for
plaque-restricted epitopes (such as malonaldehyde-modified LDL). These methods do not, however, provide any
direct information on the histological type, the predilections site, distribution, shape or the volume of the lesions.
5. Hemostasis
Hemostasis essentially depends on (i) the coagulation
and fibrinolytic systems, which mediate formation and
dissolution of plasma clots, respectively [246,247]; (ii)
platelets which are an essential component of thrombi and
contribute to thrombus formation; and (iii) the structural
integrity of the vessel wall. Defects or perturbations in any
of these mechanisms may contribute to an increased risk
for bleeding or thrombosis. Recent insights deduced from
gene-targeting experiments suggest that hemostasis during
the earliest stages of embryonic development may be less
dependent on fibrin formation and platelet function (and
more on vascular wall integrity) than anticipated, and that
fibrin formation and platelet function become progressively
more important in the control of hemostasis later during
development and after birth [1].
5.1. Thrombosis
5.1.1. Arterial thrombosis
It is somewhat surprising that the mouse has not been
more widely used as a model to test the possible therapeutic usefulness of antithrombotic agents. This may relate to
the absence (at least until recently) of reliable models to
induce arterial thrombosis in the mouse in a controllable
and quantitative manner. In addition, the small size of the
murine arteries limits the manipulation of isolated vessel
segments, a requirement of many thrombotic models in
larger species. Furthermore, the currently available experimental models of thrombosis in the mouse differ in
their extent and location (venous or arterial) of thrombus
formation.
A model of fatal thromboembolism in mice has been
developed by intravenous injection of ADP, collagen or
thrombin, alone or in conjunction with the potent vasoconstrictor epinephrine [248–250]. Thromboembolic death
results from intravascular platelet aggregation, inducing
thrombocytopenia with redistribution of platelets primarily
in the lung vasculature (as evidenced by the use of 51 Crlabeled platelets) [251]. In addition, vasoconstriction secondary to the production of thromboxane A 2 and prostaglandin F 2 a by the aggregating platelets and damaged
endothelium may also contribute to the lethality. The
resultant widespread systemic arterial thrombosis induces
respiratory distress, hindlimb paralysis and death. Typically, the number of mice with systemic thrombosis and
resultant death within a defined period, or their survival
time are compared between control and drug-treated
animals. This model is different from another pulmonary
microembolism model, based on the intravenous administration of fixation-hardened red blood cells, in which
platelets are apparently not involved [252].
Perivascularly applied electric injury (used to study
neointima formation; see above) induces the formation of
platelet-rich and fibrin-containing clots in the femoral or
carotid arteries, but their induction is less well standardized [109]. A more controllable thrombotic model
involves the topical application of a 132-mm strip of filter
paper saturated with 10% ferric chloride solution to the
adventitial surface of the surgically exposed carotid artery
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
for 3 min [253]. This induces endothelial damage, possibly
via formation of highly reactive oxidant species and
triggering tissue factor-mediated initiation of blood coagulation (specific inhibitors of the factor VIIa-tissue factor
complex block ferric chloride-induced thrombosis). A
thrombus is formed, which predominantly contains activated platelets, fibrin strands and entrapped erythrocytes.
The extent of thrombus formation is morphometrically
quantitated on evenly spaced transverse sections, using
computer-assisted planimetry. The kinetics of thrombus
formation can be indirectly monitored by measuring the
blood flow using a miniature doppler flow probe. An initial
decline in flow is followed by cyclic variations in flow
(resulting from intermittent embolization of platelet aggregates from the site of injury), evolving to complete loss of
flow. This model allows controllable and reproducible
injury as the concentration of ferric chloride, the size of the
arterial segment being injured, and the duration of injury
can be precisely controlled. In addition, complete occlusion of the carotid artery is well tolerated by mice due to
blood flow via the contralateral artery.
Transillumination by a filtered xenon lamp (wavelength
540 nm) of a surgically exposed artery after systemic
administration of a photochemical compound (Rose Bengal) damages the endothelium, initiating the formation of a
platelet-rich clot [254,255]. In order to monitor thrombosis
in larger peripheral arteries, the artery is transilluminated
by a special device containing acrylic optical fibers
positioned underneath the artery [256]. Systemic injection
of fluorescent fibrinogen improves the in situ visualization
of the formation and dissolution of fibrin strands (V. Laux,
personal communication). This model offers an opportunity to monitor the kinetics of clot formation and
dissolution in a dynamic circulation. Argon-laser irradiation of arterioles and venules in a modified dorsal skin
chamber model has been used in the rat to induce
thrombosis [257]. As dorsal skin chambers have been
miniaturized for the mouse, such a model could be adapted
for the mouse as well.
5.1.2. Venous or capillary thrombosis
A quantitative model of venous thrombosis has been
developed by pinching the femoral vein to introduce a
standardized thrombogenic injury, using the tips of a
forceps with flat circular opposing surfaces (0.1 mm
diameter; pressure of 1500 g / mm 2 ) [256]. The vessel is
transilluminated using acrylic optical fibers and clot formation (and dissolution) in the transilluminated vein can be
recorded by vital videomicroscopy and computer-assisted
image analysis. This model offers the particular advantage
of analyzing the kinetics of the formation and dissolution
of the thrombus, as well as of measuring the thrombus area
in a dynamic circulatory system.
Intraperitoneal injection of endotoxin results in systemic
activation of inflammatory cells, causing them to synthesize and release several endogenous mediators that
contribute to the pathophysiological process of septic
25
shock [258]. A procoagulant state develops after exposure
to endotoxin, largely due to the conversion of the endothelium from a thromboresistant to a thrombogenic surface
via increased tissue factor and plasminogen activator
inhibitor-1 expression, concomittant with reduced expression of urokinase-type plasminogen activator. Within 3 h
after intraperitoneal injection of endotoxin in mice, fibrin
formation is detected in glomerular and peritubular capillaries in the kidney [258]. However, the presence of fibrin
is transient, decreasing at 8 h and disappearing by 24 h.
The thrombotic response can be visualized by immunostaining for fibrin. Injection of the proinflammatory endotoxin (10 to 50 mg) in the footpad of mice induces local
inflammation and venous thrombosis, which can be quantified by counting the number of occluded vessels and their
degree of occlusion [259,260].
Clinical conditions associated with hypoxia or hyperoxia
can lead to prothrombotic diatheses. Hyperoxic lung injury
is characterized by a prominent intra-alveolar deposition of
fibrin and cell debris, the classical feature of hyaline
membrane disease, in part because of increased expression
of tissue factor (initiating blood coagulation) and PAI-1
[261]. Hypoxic stress has been implicated in the recruitment of mononuclear phagocytes and the derangement of
endothelial and monocyte anticoagulant properties (e.g. the
induction of tissue factor and the reduction of plasminogen
activator expression), all resulting in thrombus formation.
Perturbations in oxygen levels (hyperoxia or hypoxia) are
produced by placing the mice in isolated chambers with
increased (75 to 100%) or reduced (6%) oxygen for
defined periods. Fibrin deposition and platelet accumulation in the lung tissue are evaluated by immunostaining
and ultrastructural analysis (revealing the 22.5-nm strand
periodicity of fibrin), by immunoblots revealing fibringamma chain dimers, by measuring the amount of fibrin
activation products in the plasma or urine, and by deposition of 125 I-fibrin and 111 In-labeled platelets [261–263].
Another microthrombotic model relies on local cerebral
hyperthermia, regionally applied by heating the brain
surface with irrigating artificial cerebrospinal fluid in a
cranial window [264]. This induces platelet aggregation on
damaged endothelial cells, vasoconstriction and the formation of arterial thrombi. Dehydration increases the susceptibility to thrombosis in the pial microcirculation. This
model mimics many features (e.g. the microthrombi, low
platelet counts and areas of local necrosis) of the thrombotic complications found in many organs from heat stroke
victims. Thrombosis can also be photochemically induced
in the microcirculation in the exteriorized peritoneum or
cremaster muscle, and monitored by vital videomicroscopy
upon transillumination [254].
5.1.3. Transgenic models with increased thrombogenicity
Several spontaneously arisen or genetically manipulated
mutations have increased the susceptibility of mice to
thrombosis. Among them are mice with sickle cell disease
(arterial thrombosis) [265] or Lupus syndrome, sponta-
26
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
neously arisen [266,267] or acquired through passive
administration of antiphospholipid antibodies (arterial and
venous thrombosis; myocardial infarction). In addition,
transgenic mice have been generated with targeted deficiencies of the plasminogen system (e.g. of the tissuetype and urokinase-type plasminogen activators [259], or
of plasminogen [268]) or of the coagulation system (e.g. of
tissue factor pathway inhibitor [269], or protein C; unpublished observations), which induce arterial or venous
thrombosis. Mice with a targeted mutation of the coagulation factor V (factor V Leiden [270]) or of the anticoagulant thrombomodulin are at increased risk to develop
arterial or microvascular thrombosis, respectively. Mice
lacking PAI-1 (the inhibitor of t-PA and u-PA) are relatively resistant to thrombosis induced by local endotoxin
injection (footpad model) [260], by hyperoxia (lung
model) [261], by hypoxia (lung model), or by ferric
chloride application [253].
5.2. Thrombolysis
Thrombolysis can be evaluated by quantitating the
degradation of an intravenously injected 125 I-fibrin plasma
clot that becomes embolized in the pulmonary vasculature
[259,260,268]. By supplementing varying amounts of
platelets to the plasma clots, degradation of pulmonary
plasma clots that are free, poor or enriched in platelets can
be analyzed. Platelet-free pulmonary plasma clots are
spontaneously lysed within 24 h in wild-type mice. Alternatively, 99 Tcm-labeled fibrin-specific antibodies can be
included in the plasma clots [271]. Because technetium is a
strong gamma emitter, the kinetics of clot lysis in individual mice can be monitored over time by imaging mice
with a gamma camera. Clot lysis is markedly impaired in
mice lacking tissue-type plasminogen activator [259] or
plasminogen [268], or in mice overexpressing lipoprotein
(a) [271], or PAI-1 (following adenoviral gene transfer;
unpublished observations). In contrast, lysis is markedly
increased following adenoviral-mediated t-PA gene transfer
[272]. In contrast to the pulmonary clot lysis assay (which
is formed in vitro and subsequently injected into the mice),
dissolution of thrombi that are formed in situ can be
monitored in the venous pinch model (described above)
[256].
5.3. Bleeding
Acute bleeding in neonatal or adult mice is induced by
transsection of the tail 1 to 3 mm proximal to its tip,
respectively, using a sharp scalpel blade. The tail is
immersed in a recipient containing saline preheated at
378C to prevent vasospasms [273,274]. The mouse is
maintained in a horizontal position in a restrainer with the
tip of the tail 4 to 5 cm below the body plane. Bleeding is
quantitated by determining the time required until cessation of the blood stream (varying between 2 to 4 min in
normal mice). To compensate for differences in the rate of
blood flow, the amount of blood collected in the prewarmed saline recipients (or absorbed on filter devices) is
also determined by spectrophotometric analysis of extractable hemoglobin.
Delayed rebleeding, as typically occurs in patients with
deficiency of PAI-1 or a 2 -antiplasmin, can be evaluated
after transsection of the tailtip or nail cuticle, or by surgical
interventions (such as for example by removal of the
caecum) [260]. Chronic bleeding can be indirectly evaluated by determining the hemoglobin content, the number
of red blood cells and reticulocytes in the peripheral blood,
as well as by visualization of hemosiderin (reflecting
erythrocytes phagocytosed by macrophages) at the site of
bleeding.
5.3.1. Transgenic models with increased bleeding
Several transgenic mouse strains bleed spontaneously
during embryogenesis. Whereas the precise mechanisms of
bleeding in some strains remains uncertain (mice with
targeted deficiency of factor V [275], or of tissue factor
pathway inhibitor [269]), other transgenic mouse strains
bleed because of defects in the formation of the vessel
wall. This may be due to impaired endothelial assembly
(mice with targeted deficiency of TIE-1 [21], TGF-ß [111],
VCAM-1 [120], fibronectin [112], or a 4 integrin [121]), to
abnormal pericyte recruitment (mice with targeted deficiency of tissue factor [24], TIE-2 [114], angiopoietin-1
[23], PDGF-B [91]), or to abnormal smooth muscle
function (deficiency of LKLF [124]).
The birth trauma is a common cause of perinatal
bleeding, preferentially occurring in the abdomen and the
brain. Bleeding resulting from lack of proper fibrin clot
formation occurs in mice with deficiency of factor VII [25],
fibrinogen [276], factor V [275], or from absence of
platelets in NF-E2 knockout mice [277]. Mice deficient in
tissue factor pathway inhibitor [269], or protein C (unpublished observations) bleed as a result of the depletion of
coagulation factors secondary to the initiation of uncontrolled diffuse intravascular coagulation. Other mouse strains
suffer an increased bleeding tendency later in life, but only
after mechanical trauma. Some of those suffer defects in
fibrin clot formation (mice deficient in factor VIII [278],
factor IX [279,280], and mutant mouse strain RIIIS / J
[281]), or exhibit abnormal platelet function (mutant
mouse strains brachymorphic [282], subtle gray [283],
ruby-eye [284], gunmetal [285]). Other mutant mice
display increased postnatal bleeding tendency, presumably
because of vascular defects (brachymorphic, hemimelic
extra toes, ulnaless [282]), whereas other transgenic mouse
strains die because of postnatal hemorrhaging due to
abnormal vascular fragility (deficiency of fibrillin-1 [127],
collagen type I [125]). Bleeding in adult mice can be
induced by administration of anti-factor VIII / von Willebrand Factor inhibitor plasma [286].
P. Carmeliet et al. / Cardiovascular Research 39 (1998) 8 – 33
5.3.2. Gene transfer
Hemophilia B mice [279,280] are a good model to test
possible (gene)-transfer strategies. Adenovirus-mediated
transfer of human factor IX resulted in normalization of
the aPTT, and prevented bleeding. Noticeably, a marked
antibody response occurred in hemophilic CD-1 but not in
C57BL / 6 mice, suggesting that the former may be suited
to study the development of inhibitors, whereas the latter
might be better suited to study gene transfer [280]. Plasma
factor IX levels were also restored using adenovirusmediated gene transfer in another model of hemophilia B
[279]. It will be intriguing to test recombinant adenoassociated virus-mediated gene transfer of human factor IX
in these mice [287].
6. Conclusions
The availability of different inbred genetic backgrounds
and the possibility to specifically manipulate the expression of the mouse genome (via transgenesis, gene transfer,
or cell transplantation) offer a unique opportunity to
unravel the molecular basis of complex, multifactorial
biological processes and diseases such as angiogenesis,
hemostasis, atherosclerosis, and restenosis. However, the
mouse differs from the human in many ways, necessitating
careful and in depth analysis of these mouse models.
Despite species-related or model-dependent differences,
valuable insights into the molecular mechanisms can be
deduced, but extrapolation from these mouse findings to
clinical relevance in humans should be made cautiously.
Future challenges lie in the development and better characterization of such mouse models, as they will yield insights
that cannot be derived from other available animal models.
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