The Pathobiology of Intravascular Stents in Humans and Animal Models
Peter G. Anderson, DVM, PhD
Professor of Pathology
The University of Alabama at Birmingham
Birmingham, Alabama
pga@uab.edu
Introduction
Percutaneous coronary intervention (PCI) has become a frequently used clinically effective
method for the revascularization of stenosed or occluded native arteries and vein grafts. However, the
problem of restenosis continues to importantly impact the clinical utility of this procedure. Data from
retrospective and prospective studies of angioplasty, atherectomy, laser ablation, and endovascular
stenting demonstrate that with all of these interventional techniques the restenosis rate ranges from
25% to 40% (1-3). Even with the advent of drug-eluting stents, the restenosis rate varies from
approximately 8% to 35% depending on patient and lesion characteristics (4). Thus, any
revascularization technique, which necessarily involves some degree of trauma to the vessel, may lead
to restenosis in a certain cohort of patients. The specific factors that may lead to restenosis have not
been well defined. The underlying basis for restenosis involves some form of vascular trauma with
injury to endothelial and/or smooth muscle cells. Clinical and experimental studies have also shown
that restenosis may result from other injurious procedures such as electrical stimulation, freezing,
alcohol perfusion, crush injury, or physical injury produced by placement of a constricting band around
the outside of the vessel (5-7). The trophic factors, either blood born or locally produced and/or
released during these interventions, which result in active growth and phenotypic conversion of the
smooth muscle cells with exuberant extracellular matrix production after vascular injury have been
extensively studied with no clear consensus as to the specific factors responsible for restenosis (1,8-12).
It is clear that restenosis is a complex pathophysiological process, and the unraveling of the specific
factors leading to restenosis will require precise experimental design and appropriate model systems.
Various in vitro and in vivo model systems have been used to study restenosis and to investigate
methods for preventing restenosis. Intravascular irradiation after vessel injury (brachytherapy) was
initially thought to be an effective way to reduce the risk of repeated in-stent restenosis. However,
concerns have been raised regarding a late catch-up in restenosis rates, reducing its long term benefits
compared with angioplasty for in-stent restenosis (13,14). Comparisons between radiation therapy and
drug-eluting stents for the treatment of in-stent restenosis demonstrate that drug-eluting stents provide
superior clinical outcomes (15,16). The vanguard drug-eluting stents that were first approved by the
FDA had somewhat different mechanisms of action. That both paclitaxel and sirolimus inhibit but do not
prevent restenosis indicate that renarrowing after arterial injury is a complex process (10) the exact
etiology of which continues to elude investigators. Results from these studies have elucidated many
mechanisms involved in the restenosis process; however, there are some discrepancies in the efficacy of
treatment protocols when comparative studies are performed on different animal species, including
man. Thus, there appear to be significant species differences in response to vascular injury that must be
considered when performing and evaluating studies of restenosis (17-19).
Mechanical Vascular Injury: Experimental Models and Study Paradigms
The choice of model systems for studies of vascular biology and specifically studies of restenosis
requires clear identification of the experimental endpoint. It must be acknowledged from the outset
that all model systems have limitations and that these experimental systems are just models of a
process that occurs in humans. Thus, no experimental system can completely and accurately
recapitulate the restenosis phenomena seen in an atherosclerotic coronary artery of human patients.
However, it is possible by careful experimental design to answer specific questions related to the
phenomena of restenosis using in vitro systems or animal models (20).
In vitro systems can be very precisely controlled and specific factors or processes can be isolated
and studied (21-25). If a specific hypothesis is to be tested, in vitro systems can be manipulated in such
a way that only one variable is examined. These studies are useful in studies of the cell and molecular
biology of endothelial cells or smooth muscle cells. Although beneficial in basic scientific investigation,
in vitro studies cannot be used to mimic or recapitulate the restenosis process in vivo. The procedures
used to isolate smooth muscle cells or endothelial cells and the techniques used to maintain them in
culture are far removed from the milieu of an atherosclerotic coronary artery in a patient. Also, with in
vitro techniques it is impossible to maintain the interaction that occurs between the various cell types
and the blood born factors that impact of the restenosis process in vivo. Studies of smooth muscle cells
in culture demonstrate that after passage and after transition from the contractile to the secretary
phenotype, key regulatory signaling pathways are altered or lost (26-28). These findings suggest that
using cultured or passaged smooth muscle cells in studies of the vascular response to injury may be
problematic. Thus, data from in vitro studies should be used to evaluate specific mechanisms of the cell
and molecular biology of endothelial cell and smooth muscle cells, not to make definitive conclusions
about the restenosis process in vivo.
Investigators have utilized various animal species, different arteries or veins, and numerous
types of interventions to induce restenosis. These model systems fall into three basic categories. In one
group, models are designed to produce a very reproducible neointimal reaction that can be easily
quantitated. Examples of this type of model system include the rat aortic or carotid artery balloon injury
model and rabbit models of femoral or iliac artery injury. In these systems the degree of restenosis is
predictable and uniform. Thus, experiments designed to modify neointimal growth can be easily tested
in these models. The second type of model system often utilizes coronary arteries in larger animals with
artery sizes similar to man. These model systems also endeavor to produce a consistent neointimal
proliferative reaction; however, the reproducibility and ease of performing these experiments may be
somewhat compromised in order to obtain the benefits of using a larger animal model. In these models
injury is produced in normal arteries and the response to that injury is evaluated. Examples of models in
this group include rabbit or pig coronary and carotid arteries. These models will be discussed in detail
below. The third type of model system used to study restenosis attempts to recapitulate the conditions
seen in human atherosclerotic coronary arteries. These systems utilize artery injury and atherosclerotic
diets to induce atherosclerotic lesions in arteries. After development of atherosclerotic lesions,
mechanical interventions are performed (e.g. angioplasty, stents, atherectomy) and the response of the
diseased artery to this intervention is evaluated. These types of model systems are good for testing new
equipment or new techniques. These models are also useful for evaluating the response of the diseased
artery to these insults and the ability of therapeutic techniques to diminish the restenosis phenomena.
Unfortunately, the complexity of these model systems makes it very difficult to obtain reproducible
injury or reproducible restenosis; thus, statistical evaluation of the efficacy of therapeutic techniques is
very difficult. In essence, trying to recapitulate human atherosclerosis makes these models so complex
that these experiments have many of the same problems as human trials, i.e. large variability and large
numbers of subjects needed to reach statistically significant end points. Examples of these types of
animal models are femoral or iliac artery injury plus atherosclerotic diet in rabbits, injury plus
atherosclerotic diet in minipigs, and atherosclerosis in nonhuman primates. These models have distinct
advantages and disadvantages that will be discussed below.
The primary factor to be considered when choosing animal models of restenosis is the endpoint
for the experiment. Each animal model has strengths and weaknesses that must be factored into the
study plan. Each animal species has a slightly different anatomy and physiology, and these differences
may or may not impact the experimental results when compared to man. Thus, the model system
should be carefully evaluated and pilot studies should be performed prior to undertaking a large study.
Animal Models in the Study of Vascular Interventions
Numerous animal models are currently being used in vascular biology research. This review will
briefly discuss some of the more commonly used models used to investigate the pathogenesis of
intravascular stents and restenosis and we will describe pros and cons of each model system.
Rabbits. Rabbits have been used extensively to study the pathogenic mechanisms of restenosis (29-34).
Rabbits are relatively inexpensive to procure and maintain and they lend themselves well to laboratory
experiments. Most studies have utilized the femoral or iliac arteries; however, studies of the carotid
arteries and aorta have also been performed. Of these vessels, only the femoral artery is a muscular
artery. After vascular injury rabbits develop a neointimal proliferative response that is uniform and
reproducible, and as with rats, is composed primarily of smooth muscle cells. One advantage of the
rabbit model over the rat is that the femoral and iliac arteries of an adult rabbit are similar in size to
coronary arteries in man (30). Rabbits on a normal diet do not develop naturally occurring
atherosclerotic lesions; however, with a high cholesterol diet, serum cholesterol levels of greater than
2,000 mg/dl can be achieved. In these hyperlipemic animals, lipid laden macrophages (foam cells) are
deposited in the media and intima of large arteries as well as in the parenchyma of the spleen, liver, and
lymph nodes. This vascular foam cell deposition does not resemble the fibrocalcific atherosclerotic
lesions in man. Other model systems in rabbits utilize a moderately high cholesterol diet and some form
of vessel injury, e.g. air-drying or balloon injury (31, 32). In these models the arterial lesions consist of
foam cells in the media and intima as well as a smooth muscle cell proliferative response similar to the
type of response seen in human atherosclerotic arteries that have undergone balloon injury. This model
system does not usually have the well-developed atheromatous lesion with a fibrous cap or areas of
calcification that is seen in human arteries; however, the major components of the atherosclerotic
process are present in the rabbit atherosclerotic model. A third rabbit model that may be used in
restenosis research is the Watanabe rabbit. This strain of rabbits has an inherited deficiency in lowdensity lipoprotein receptors and the resultant hyperlipidemia predisposes the rabbits to atherosclerotic
vascular disease (34). The pattern of atherosclerosis is similar to that found in patients with familial
hyperlipidemia. Well-developed vascular lesions contain a cholesterol-filled necrotic core with areas of
calcification and a fibrous cap. Some studies have utilized heterozygous Watanabe rabbits fed a high
cholesterol diet (33). This model may have advantages in that the morphology of the lesions more
closely resembles human coronary artery disease as opposed to the foam cell lesions seen in normal
rabbits fed high cholesterol diets.
Non-atherosclerotic rabbits have been use in studies of restenosis; however, many studies have
involved some experimental manipulation to induce atherosclerosis. Even though the atherosclerotic
lesion in rabbits is not identical to human atherosclerosis, there is intimal thickening, lipid accumulation
(primarily foam cells) and fibrosis within the vessel wall, and some atrophy or degeneration of the media
with adventitial thickening by fibrosis. Thus, any additional intervention (angioplasty, stent placement,
or atherectomy) is performed in a diseased artery not a normal artery. This in itself may help to give
credence to this model system as opposed to models where injurious interventions are performed on
normal arteries. The atherosclerotic rabbit model is very good for investigations of new angioplasty
balloons, intravascular stents, and atherectomy devices. Evaluation of new devices and accurate
quantitation of the degree of restenosis can be accurately evaluated in large numbers of animals.
Swine. Swine models of vascular injury and restenosis are considered by most investigators to be the
gold standard animal model for studies of vascular intervention. Pigs are relatively inexpensive to
procure, are readily available, and are well suited for vascular research (35-37). Juvenile farm pigs can
be utilized for acute and short-term experiments of vascular injury. However, farm pigs grow rapidly
and can reach an adult weight of greater than 400 kg. Thus, all studies in farm pigs must necessarily be
of short duration. This problem of extreme size in pig models can be overcome by the use of specially
bred mini- or micropigs (38). These animals reach an adult weight of 30 to 40 kg. These animals are
available commercially; however, the procurement costs are four to five times greater than farm pigs.
This increased procurement cost is outweighed by the utility of using these specially bred laboratory
animals, the decreased cost of maintaining these animals, and the ability to perform long term
experiments with these animals. Adult farm pigs develop naturally occurring atherosclerotic lesions
(39). Also, in both farm pigs and minipigs, high cholesterol diets will produce an elevation in serum
cholesterol and increased low density lipoproteins. These serum lipid changes predispose to
atherosclerosis. The lesions seen in the naturally occurring atherosclerosis closely resemble human
atherosclerotic lesion, including lipid deposition, calcification, and development of a fibrous cap (38, 40,
41). Lesions from experimental models of atherosclerosis have some characteristics similar to man;
however the overall character of the lesions demonstrate little similarity to those commonly seen in
human autopsy cases (42, 43).
Studies of balloon injury in pig coronary arteries involves overstretching and injury to the vessel
wall (9,20,44). The type of injury and the restenotic process is quite dissimilar to the classic rat carotid
balloon injury model. In the rat the vessel is denuded of endothelium and there is little or mild medial
injury. However, in the pig coronary artery model there must be significant medial injury before you
elicit a restenotic reaction. Since this model involves injury to a normal vessel, the type of lesion caused
by the angioplasty balloon or the stent is somewhat different than what one sees after interventional
procedures in human atherosclerotic vessels. In man it goes without saying that interventions are not
usually performed unless there are underlying atherosclerotic lesions in the vessel. Work from our
laboratory and others (45, 46) have demonstrated that in atherosclerotic vessels balloon injury usually
causes laceration at the “shoulder region” of the atherosclerotic plaque with dissection between the
plaque and media or the adventitia. In normal pig coronary arteries there are no atherosclerotic
plaques. Thus the angioplasty balloon stretches the vessel until there is laceration of the media with
stretching of the external elastic lamina and the adventitia. Immediately after the balloon injury a layer
of platelets and thrombus form on the exposed external elastic lamina. Examination of vessel segments
immediately after overstretch balloon injury demonstrate the laceration of the media and the exposed
external elastic lamina. Within four days after the injury there is migration of smooth muscle cells into
the thrombus that forms in the rent produced by the medial laceration. Over time the space produced
by the overstretch injury is filled in as neointima forms. Studies from our laboratory have shown that
most of the neointima forms by 14 days post injury with only a moderate increase in neointima between
14 and 28 days (44).
The response of pig coronary arteries to intravascular stenting in normal and atherosclerotic
swine has been well characterized (18, 20, 47, 48). In these studies vascular injury is induced by
placement of wire stents which are deployed by an angioplasty balloon, thus producing endothelial
denudation and stretch injury to the vessel wall. Stents and delivery balloons can be sized such that the
size of the fully inflated balloon inside the vessel produces varying degrees of vessel wall stretch injury.
In some studies the balloons are purposely over stretched (50 to 100% over sizing, e.g. 3.0 mm diameter
stent inside a 1.5 to 2.0 mm diameter vessel) to produce significant neointimal proliferative reaction. It
is also possible to use balloons and stents that produce less vessel wall injury (10 - 20 % over sizing)
which produces less neointimal reaction (20, 47).
In our studies of the severe overstretching model of stent placement in the pig (48, 49), we have
observed frequent laceration of the internal elastic membrane with stent wires being embedded into
the media or adventitia. Despite this degree of vessel injury, only one out of 45 pigs demonstrated very
mild extravasation of blood that was clinically insignificant. In pigs that were sacrificed twenty eight
days after stent implantation there was a moderate thickening of the adventitia around the stented
vessel segment and the degree of neointimal and adventitial reaction is clearly visible. The primary
reaction within the vessel wall consisted of neointimal tissue which was found overlying the stent wires
where the vessel wall had been indented and to a lesser extent around the entire circumference of the
vessel. The neointimal tissue consisted of smooth muscle cells with varying amounts of eosinophilic
extracellular material and occasional small blood vessels. Morphologically, the neointimal tissue is
similar to human restenotic tissue seen after angioplasty or stenting (20, 45). In vessels where the stent
wires were embedded deep into the media or into the adventitia, there is often a chronic inflammatory
reaction consisting of macrophages, lymphocytes, plasma cells and occasional eosinophils.
A significant neointimal proliferative reaction is seen in the oversized stent coronary artery
model in the pig (20, 48, 49). This neointimal reaction is morphologically similar to the reaction seen in
humans after angioplasty; however, the degree of proliferation may be greater than what is usually seen
in man. This exuberant proliferative reaction may be so intense that pharmacologic means of
preventing restenosis would be unable to ameliorate the neointimal proliferative response. Thus,
studies utilizing compounds or techniques that may have promise in preventing restenosis in man could
give a negative result in the pig.
Investigations of restenosis in conjunction with atherosclerosis in pigs have utilized minipigs for
these long term studies. In one study (50) minipigs were placed on an atherogenic diet consisting of 2%
cholesterol, 15% fat, and 1.5% sodium cholate two weeks prior to balloon denudation of the coronary
artery. Four to 5 months after the original vascular intervention, atherosclerotic lesions had developed
at the area of denudation. These lesions were then used as sites for stent implantation in order to
evaluate the response of a diseased artery to stent implantation. At sacrifice there was a significant
neointimal proliferative reaction within the stented region of the vessel. The character of these lesions
was similar to what was seen in the stented coronary arteries of pigs not on the atherogenic diet
(described above), except that these arteries also contained foam cells and more fibrous connective
tissue within the neointima. In additional studies with minipigs, stents were implanted in normal
coronary arteries 2 weeks after the pigs had been started on an atherogenic diet (51). In these studies
the character of the neointimal proliferation was similar to the non-atherogenic stented arteries
described above and no mention was made of foam cells in these lesions.
It is apparent that swine models of vascular injury and restenosis are useful experimental tools
for studies of the pathogenesis of restenosis. The neointimal proliferative response is very similar in
character to that seen in man; however, as mentioned above, the proliferative response is very intense
and may be more severe than the proliferative response seen in man. In addition it is not apparent at
this time what role the stent wire plays in producing the neointimal reaction seen in these animals.
Further studies to characterize this restenosis reaction are needed to better determine the utility of
these experimental systems as models for restenosis in man.
Intravascular Stents: Clinical and Experimental Applications and Characteristics
In-stent restenosis is a complex process, involving vascular remodeling, elastic recoil, smooth muscle cell
migration and proliferation, extracellular matrix production, and platelet activation. Although stent
design and materials have significantly improved, nearly eliminating issues of vascular elastic recoil and
contraction at the site of stent deployment, in-stent restenosis persists. Current efforts to improve
stents have focused on stent designs to influence shear and wall stresses, thinner stent struts, materials
with greater tissue compatibility, and methods to accelerate endothelial coverage after stent
deployment.
Stent Design. Stents deployed in coronary and peripheral arterial vessels are designed to be either
balloon-expandable or self-expanding. Those stents that are balloon-expandable are primarily used in
the coronary or renal arteries, whereas self-expanding stents more often are used in peripheral vessels.
Self-expanding stents have shape-memory, and return to their expanded shape after undergoing
compression or flexion. These stents are primarily placed in locations such as carotid and superficial
femoral arteries, which undergo significant bending and compression. Balloon-expandable stents are
used more in locations that require greater radial support, such as at the ostium of a vessel. Stent
design itself significantly influences the rate of restenosis and thrombosis after deployment (52).
Factors appear to include the number and method of connection of the stent struts, the degree of stent
recoil after deployment, strut thickness and shape, and the degree of scaffolding of the artery by the
strut distribution (53-56). There is general consensus that stents with less recoil and with thinner stent
struts are associated with less restenosis.
Stent design and location of deployment significantly influences the stresses on the vessel wall itself,
further influencing in-stent restenosis. Wall shear stress (WSS) is the “frictional” force exerted by the
viscosity of the circulating blood on the intimal layer of a vessel (57). WSS is influenced by vessel
geometry, such as curvature and branching, with regions of low wall shear stress associated with
accelerated atherosclerotic plaque development (58, 59). Stent deployment creates regions of
decreased WSS, which are associated with increased neointimal proliferation and greater intimal
thickness (60, 61). Regions of low WSS are influenced by strut thickness, number of stent struts, angle
between struts, strut width, and arterial surface area covered by stent (62).
Another form of stress affecting the arterial wall is the perpendicular component of stress, associated
with outward pressure such as from blood pressure. This is called wall normal stress. After a stent is
deployed, this outward normal stress varies with stent design. Greater wall normal stress has been
associated with increased atherosclerosis and neointimal proliferation (63). In addition the stiffness of a
stent placed in a curved vessel causes arterial straightening. There may be a three-fold difference in
stiffness between stent types (64). This straightening, and resultant greater curvature at the ends of the
stents, appears to alter shear stress and influence restenosis (65).
Stent materials. When initially FDA approved, stents were created from 316L stainless steel. With the
development of multiple stents, other metals have been used, most commonly Nitinol (nickel-titanium),
and cobalt alloy (cobalt-chromium). Although other materials continue to be used, balloon-expandable
stents are made primarily of stainless steel and cobalt alloy; whereas self-expanding stents are primarily
of Nitinol. Once a stent is deployed, the surface rapidly is covered with a monolayer of soluble proteins,
binding based on surface affinity and plasma concentration. The proteins that bind, such as fibrinogen
and albumin, play a key role in the cellular response to stent and platelet adhesion and activation (6667). The stent materials used and their surface treatments significantly influence protein adhesion and
cellular response (68, 69).
Corrosion. All metals are subject to corrosion from their local environment, and blood is considered
extremely corrosive to metallic materials (70). During production, stents undergo surface treatments
such as electropolishing, to create a uniform oxide layer and improve corrosion resistance (71, 72). The
durability and ability to repassivate or repair the surface layer varies based on metal type and treatment
used, and once there is a break in the coating, there is increased risk of corrosion. Release of ions from
implanted metallic materials into local tissues has been associated with cytotoxicity in vitro, and has
been studied extensively in the orthopedic and dental literature (73). Corrosion products of Nitinol
stent wire can cause smooth muscle cell necrosis, altered cell morphology, and decrease cell numbers.
Likewise, stainless steel corrosion products, particularly nickel, may change smooth muscle cell
morphology and induce cell necrosis. Low levels of metallic ions may be associated with
proinflammatory activity, such as release of IL-6. In addition, the presence of shear stress may influence
a local inflammatory response. Limited in vivo and clinical trials data exists, evaluating the role of surface
coatings and ion effects. Stainless steel stents coated with gold have increased restenosis rates
compared with those without coating, suggesting the influence of local ion effects (73-75). In addition,
improving electrochemical properties by the use of a titanium-nitride-oxide coating on a stainless steel
stent, has demonstrated decreased platelet adhesion, fibrinogen binding, and clinical in-stent restenosis
(76).
Analyses of stents obtained post-mortem indicate that stent corrosion does occur in humans, with
measurable release of ions into surrounding tissues (77-79). Evaluation of metallic coronary and
peripheral endovascular stents post in vivo implantation revealed evidence of electrochemical and
mechanically-induced corrosion. Vascular tissue surrounding corroded stents was shown to
experience transfer of metallic elements. These results, although from a limited number of specimens,
encourage further investigation of the effect of in vivo corrosion on the structural integrity of stents
and its impact on in-stent restenosis. New trends in stent design, materials, and coatings portend
better products for human use (80). These new technologies include bioabsorbable scaffolds that are
designed to serve the mechanical purpose of a stent and then dissolve away when no longer needed.
This idea, although not new, is gaining headway in clinical use (81).
Concluding Remarks
Cardiovascular disease is an increasingly important cause of morbidity and mortality in the
developed world and a thorough understanding of the response of blood vessels to injury is crucial for
development of effective prevention and treatment approaches. Blood vessels are dynamic structures
that respond in fairly predictable ways to chemical, infectious, and mechanical injury. Connective tissue
cells and the extracellular matrix produced by synthetic smooth muscle cells also play a role in the
response of blood vessels to injury. Blood constituents, particularly platelets and components of the
coagulation cascade, are also primary participants in vascular injury and repair. The response to injury is
somewhat limited whereas the mechanisms of injury are numerous, complex, and often poorly
understood. Thus, characterization of the morphologic changes often provide important directions for
investigating the nature and mechanisms of injury. It is therefore essential that pathologists along with
vascular biologists provide as accurate as possible an anatomic/morphologic description of the changes
observed.
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