Chapter 13
ANTIOXIDANTS AND RESTENOSIS AFTER PERCUTANEOUS CORONARY INTERVENTION:
ANIMAL STUDIES
Eric Durand, Ayman Al Haj Zen, Camille Brasselet, Antoine Lafont
European Georges Pompidou Hospital and INSERM EOO-16, Faculte de Medecine Necker- Enfants Malades, Paris, France
Introduction
Restenosis remains the principal limitation of percutaneous coronary intervention (PCI). We have learned from animal studies that constrictive remodeling is the principal mechanism of restenosis after balloon angioplasty^^'^l In contrast, in-stent restenosis is related to neointimal hyperplasia^^l These data were confirmed in humans by intravascular ultrasound (IVUS)^^'^\ After balloon arterial injury, oxidative stress is increased and contributes to endothelial dysfunction, macrophage activation, and release of cytokines and growth factors^^^\ In the past decade, several antioxidants have been evaluated in various animal models after balloon angioplasty. Probucol and vitamins (E with or without C) effectively reduced restenosis by promoting favorable remodeling (i.e., enlargement remodeling). Moreover, these treatments decreased neointimal hyperplasia which is the target for in-stent restenosis. In an era of nearly 100% stent implantation, it is now time to evaluate their efficacy in animal models of in- stent restenosis after either systemic administration or local delivery.
Mechanisms of restenosis after balloon or stent angioplasty
In the 1980s, neointimal hyperplasia was considered the principal mechanism of restenosis. We have learned from animal models in the 1990s that neointimal hyperplasia is not the principal target of restenosis (i.e., lumen loss at the site of angioplasty) after balloon angioplasty. Indeed, there
was no correlation between restenosis and severity of neointimal hyperplasia^^'^\ In contrast, restenosis was closely correlated with arterial remodeling in rabbit, pig, and non-human primate models^^"^\ Schematically, constrictive remodeling is observed in the presence of restenosis, and enlargement remodeling in its absence. Interestingly, these findings were confirmed by IVUS in humans^^\ During the same period, we learned from two clinical trials that stents reduce the incidence of restenosis by about 30%
as compared to balloon angioplasty^^^'^^\ In animal models and in humans, it has been clearly demonstrated that neointimal hyperplasia is the principal mechanism of in-stent restenosis after PCI^'^'^l Indeed, the severity of in-stent restenosis is closely correlated with the extent of neointimal hyperplasia^^\
Neointimal hyperplasia is related to the severity of injury, smooth muscle cell proliferation, collagen synthesis and inflammation^^^'^^^
Antioxidants and restenosis after balloon angioplasty
The methodology and results of these experimental studies are summarized in tables 1 and 2. First, it is surprising to note that none of these studies evaluated the amount of reactive oxygen species (ROS) produced after angioplasty as well as the effect of antioxidants on ROS production.
However, in 1997, Nunes et al showed that ROS production increased after balloon injury, and decreased with antioxidants^^^\ The principal source of ROS was recently attributed to NAD(P)H oxidases after vascular injury^^^'^^\
In the 1990s, a large number of studies evaluated the effect of probucol after arterial injury in rat, pig, and rabbit models {Table 1, 17-23). Probucol was administrated before angioplasty (from two hours to two weeks) and was continued during two or four weeks^^^"^^\ In these studies, neointimal hyperplasia was significantly reduced as compared to control animals^^^"^^\
However, these authors neither evaluated restenosis nor arterial remodeling^^^"^^\
Similarly, the effect of vitamin E with or without vitamin C was evaluated in cholesterol fed rabbit, rat and swine models (Table 2, 24-27).
With a pretreatment (from one to nine weeks) and a treatment duration of one to three weeks after balloon angioplasty, all studies (except Nunes et at^) observed that neointimal hyperplasia was reduced as compared to control animals^^'^'^^l Only two studies evaluated the effect on restenosis and one the effect on remodeling^^'^'^^^ Vitamin E or the combination of vitamins C and E reduced restenosis by promoting favorable remodeling^^^l
Antioxidants and restenosis after PCI: animal studies 329
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Table 2, Vitamins C and £ in animal studies
^ ^ - - - ^ Model
Artery
Animal number Pretreatment Treatment duration Double injury Type of injury Angiography Restenosis Morphometry 1 Lumen area 1 Intimal 1 hyperplasia 1 Arterial 1 remodeling
Lafont et al (25)
Rabbit Femoral
29 3 weeks (vitamin E)
3 weeks Yes Air desiccation+
balloon injury Yes
i
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t
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Nunes et al (24) Swine Coronary
44 1 week (vitamins
E+C) 2 weeks
No Balloon angioplasty
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t
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t
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1 week No Fogarty denudation
ND ND Yes ND
i
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Chen et al (27) 1 Rabbit
Aorta 96 2 weeks (vitamin E)
3 weeks No Fogarty denudation
ND ND Yes ND
i
ND
ND: not done, NS: not significant
Interestingly, similar results were reported in humans. The Multivitamin Probucol (MVP) trial evaluated by angiography and IVUS the effect of vitamins C and E or probucol on restenosis after balloon angioplasty^^^'^^\ In summary, they confirmed that probucol, but not vitamins C and E, reduced restenosis^^^\ This beneficial effect was attributed to a favorable effect on arterial remodeling^^^^
The effect of adenovirus-mediated gene transfer of superoxide dismutase (SOD) with or without catalase was evaluated after balloon injury in the rabbit model^^^' ^^\ ROS production was decreased as compared to control animals. After adenovirus-mediated gene transfer of SOD and catalase, neointimal hyperplasia and constrictive remodeling were significantly reduced and resulted in less restenosis^^^'^^\
Antioxidants and restenosis after PCI: animal studies 331 In conclusion, antioxidants reduced both neointimal hyperplasia and
constrictive remodeling resulting in less restenosis after balloon injury. The beneficial effect on restenosis is limited to the remodeling process since the extent of neointimal hyperplasia is not related to restenosis^^'^l
The next step is to understand how it works (i.e., how antioxidants promote favorable remodeling after balloon injury). Vessel remodeling after arterial injury is a complex healing process. Endothelial dysfunction, collagen accumulation, and inflammation are probably the three principal mechanisms of constrictive remodeling after balloon angioplasty^^^'^^\ After angioplasty, some endothelium is removed, and preserved adjacent endothelial cells migrate and proliferate to reline the vascular wall. Arteries with regenerated endothelium may exhibit impaired endothelium-dependent relaxation via reduction of nitric oxide (NO) production^'^^'^'^^ Interestingly, the severity of endothelial dysfunction is correlated with restenosis and with constrictive remodeling^^^\ The mechanisms controlling re-endothelialization after angioplasty are not well established. Re-endothelialization does not seem to be mediated by NO since L-arginine and L-NAME have no effects on the re-endothelialization process after arterial balloon injury^^^l Recently, Lau et al elegantly demonstrated that probucol accelerates re- endothelialization and improves endothelium-dependent relaxation after balloon injury^^^\ Interestingly, we also observed that adenovirus-mediated gene transfer of SOD and catalase accelerates re-endothelialization and improves endothelium-dependent response to acetylcholine^^^\ The more effective re-endothelialization may be explained in part by the fact that antioxidants are known to inhibit endothelial cell apoptosis after angioplasty^^^'^^l In turn, improved re-endothelialization might contribute to the reduction of neointimal medial growth observed in probucol-treated animals. Therefore, the beneficial effect of antioxidants on remodeling after balloon injury may be mediated via an acceleration of functional re- endothelialization.
Collagen accumulation is the second determinant of constrictive remodeling after balloon angioplasty. Indeed, we and others showed that collagen increases after angioplasty, and that this increase correlates with severity of restenosis and constrictive remodeling^^^\ The interaction between antioxidant therapies and collagen content has recently been evaluated after balloon injury. Antioxidants decrease collagen content after arterial injury but it is not known whether they interact with collagen synthesis or degradation^^^I However, it has been shown in other experimental models that oxidative stress increases collagen synthesis and decreases metalloproteinase activity^^^'^^\
Finally, inflammatory cell infiltration is the third determinant of arterial remodeling via production of cytokines and growth factors. It has been
shown that antioxidant therapies reduce monocyte-macrophage infiltration after balloon injury^^^'^^\ Therefore the beneficial effect of antioxidant therapies on remodeling after balloon injury may be mediated by an improvement of functional re-endothelialization, and a reduction of collagen content and inflammation.
Antioxidants and in-stent restenosis
As described above, the principal mechanism of in-stent restenosis is neointimal hyperplasia. It is logical to hypothesize that antioxidants are capable of preventing in-stent restenosis since it has been demonstrated that probucol and vitamins reduce neointimal hyperplasia after balloon injury^^^"
^^\ However, to our knowledge, antioxidants have been poorly evaluated in animal models of in-stent restenosis. Surprisingly, only one negative study has been published concerning the effect of probucol on restenosis after stent angioplasty in animal models^'^^^ Probucol was administrated one week before stent implantation in porcine coronary arteries^'^^^ The authors did not find any positive effect by IVUS. Indeed, restenosis and neointimal hyperplasia were similar in the probucol and control group^'^^^ In humans, the effect of probucol after stent angioplasty were evaluated in the Canadian Antioxidant Restenosis Trial (CART-1) pilot study^'^^^ Patients were treated for two weeks before and four weeks after PCI. As compared to placebo, probucol significantly reduced in-stent restenosis. This study was also designed to evaluate the best dose of AGI-1067, a metabolically stable modification of probucol, for a larger trial, the Canadian Atherosclerosis and Restenosis Trial (CART-2). This trial was mandatory since it is known that probucol induces a prolongation of the QT interval which remains a long- term safety concern. This new metabolite does not increase the QT interval.
To our knowledge, AGI-1067 has never been tested in animal models of in- stent restenosis. In human, AGI-1067 with a pretreatment period of two weeks, and a treatment duration of four weeks, reduced in-stent restenosis through a favorable effect on neointimal hyperplasia. A randomized multicenter study is therefore conducted (CART-2) to evaluate the effect of AGI-1067 with a treatment duration of six weeks in a larger population. One can regret that preclinical studies were not published or performed prior to this study.
Conclusions
Antioxidants therapies reduce constrictive remodeling and restenosis after experimental balloon injury. These favorable results led to the MVP trial which clearly demonstrated that probucol reduces restenosis after
Antioxidants and restenosis after PCI: animal studies 333 balloon angioplasty in humans. In contrast, antioxidants have been poorly
evalued in the setting of in-stent restenosis in animal models. However, promising premilinary results have been obtained in humans with AGI-1067, a metabolically stable modification of probucol. However, we must keep in mind that systemic antioxidant therapies need a pre-treatment period of at least two weeks before angioplasty, which is not available in the setting of PCI during acute coronary syndromes.
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