A major challenge in the field of cardiovascular therapeutics is the discovery of a drug that is capable of preventing restenosis. To date, unfortunately, all pharmacological strategies for limiting restenosis have fallen short of the goal. Crucial to the successful discovery of an efficacious agent is the understanding of the mechanisms responsible for restenosis in humans. However, despite enormous research efforts, the exact pathological processes responsible for the disease in humans are not well understood, and animal models have not been able to simulate the complexity of the clinical situation. Indeed, none of the drugs that have been shown to limit restenosis in animal models have yielded positive results in human clinical trials. On the basis of animal experimentation and pathological examination of human restenotic lesions, a major hypothesis for the development of restenosis involves VSMC migration and proliferation. Thus, a popular strategy for therapeutic discovery is to use one of the animal models of neointimal hyperplasia and to examine the effects of specific drugs designed to block the processes of VSMC migration and/or proliferation. Multiple biologically active mediators have been shown to participate in these processes. Among these, Ang II is of particular interest, because pharmacological inhibitors of Ang II production or action are now clinically available. In cell culture, Ang II has been shown to stimulate growth of VSMCs isolated from several species such as rats, rabbits, and humans.1 2 3 4 More importantly, in vivo infusion of Ang II significantly increases DNA synthesis in normal, uninjured arteries and enhances neointimal development in the rat carotid artery after balloon injury.5 Moreover, using in vivo gene transfer, we have demonstrated that overexpression of the ACE gene in the rat carotid artery increased DNA synthesis and induced vascular structural changes as the result of the increased local generation of Ang II.6
Several reports have demonstrated the ability of ACE inhibitors to block neointimal development in the rat, guinea pig, and rabbit.7 8 9 The actions of the ACE inhibitors are due in part to the decrease in Ang II production, since the effects can be mimicked by the AT1 receptor antagonists.10 The accumulation of kinins may also play a role, since kinin receptor antagonists have been shown to partially attenuate the protective effects of the ACE inhibitors in the rat carotid artery injury model.11 The effect of kinins appears to be mediated in part by the kinin-dependent increase in nitric oxide production.
Early enthusiasm for the potential use of ACE inhibitors as a therapy for restenosis has diminished because other investigators soon demonstrated species differences in the ability of these agents to block neointimal hyperplasia. Indeed, attempts to duplicate the rat, rabbit, and guinea pig results in the pig12 and baboon13 models have failed. More importantly, the human trials of cilazapril on restenosis (MERCATOR and MARCATOR) yielded negative results.14 15 In this issue of Circulation, Huckle and colleagues15A have used angiotensin receptor antagonists in an attempt to block neointimal development after balloon dilation and stenting of the porcine coronary artery. In their studies, AT1 receptor subtype–selective antagonist administration did not block lesion formation, resulting in the conclusion that in this porcine model, unlike the balloon-injured rat carotid artery model, Ang II may not play a role in the development of the neointima.
How can one explain the discrepancies among the studies? Are we to assume that there are fundamental differences among the species in the cellular responses to angiotensin? Or is it more likely that differences exist in the in vivo biological response to injury? Perhaps a more important question concerns the predictability of any animal model for human response in clinical studies. The Table⇓ summarizes the possible explanation for the species-differential responses to renin-angiotensin blockade on neointimal hyperplasia. The list underscores the many variables that determine the outcome of any pharmacological intervention in different experimental models of restenosis. Indeed, these determinants also influence the outcome of clinical trials. For example, it has been proposed that the negative outcome of the MERCATOR and MARCATOR trials was due to inappropriate dose of cilazapril, protocol for drug administration, and/or species-specific differences.16
With respect to the differences observed among species with blockade of the renin-angiotensin system, one may ask whether the fundamental cellular responses to Ang II in terms of growth promotion, migration stimulation, or matrix elaboration differ between species. Previous studies have shown that Ang II can promote the growth of cultured SMCs derived from various animal species. The potency of the growth response may be variable, but is this due to an actual difference in SMCs between species or to differences influenced by culture conditions? Even in cultured SMCs from the same species, eg, the rat, differences in the growth response to Ang II (ie, hypertrophy versus hyperplasia) related to animal strain, age, and culture condition have been observed.17 In this context, it should be pointed out that the effects of Ang II on porcine coronary VSMC migration and growth was not determined in the study by Huckle et al.15A
In light of the potential caveats to cell culture studies, perhaps it is better to ask whether Ang II in vivo will induce structural changes in the vasculature. As mentioned above, Ang II infusion increases DNA synthesis and induces vascular remodeling in both injured and normal rat arteries. However, the in vivo growth response to Ang II infusion in other species is unknown. An in vivo study to evaluate the ability of Ang II infusion to alter vascular structure in these other models would be relatively simple and might provide insight into the determinants responsible for the vascular responses to Ang II in these species.
Differences observed among species in the ability of inhibitors of the renin-angiotensin system to alter the natural course of vascular injury may also be due to differences in the pathobiological responses to injury, ie, the degree of activation of cytokines, growth factors, and specifically, the renin-angiotensin system. These pathobiological responses are influenced by the complexity of the model used, and it is highly likely that as the complexity of the model increases, the relative contribution of any single agent to the growth response decreases. Thus, in this porcine coronary artery model, it would be useful to examine whether tissue ACE, angiotensinogen, or other components of the renin-angiotensin system behave differently from those of rat arteries in response to injury.
Another important consideration for the role of the renin-angiotensin system is that this system may be far more complex than is generally appreciated. For example, Ang II synthesis occurs both in the circulation and within tissue sites and, pertinent to this discussion, its production in tissue has been reported to be increased after vascular injury.16 Furthermore, the contribution of kinins to the overall vascular protective effects of ACE inhibitors may complicate the interpretation of these drugs. In addition, recent data suggest that in several species, including humans (but not rats), Ang II production may be mediated by enzymes other than ACE (such as human chymase18 ), suggesting that the Ang II synthetic pathway may differ among species. The local production of Ang II via both ACE-dependent and ACE-independent mechanisms would complicate an evaluation of ACE inhibitor action. AT1 receptor antagonists can block Ang II action irrespective of the synthetic pathway(s) of Ang II in the blood vessel. However, the existence of multiple Ang II receptors complicates the action of AT1 receptor antagonists. Could differences in AT2 receptor expression and function play a role in the species-specific responses to renin-angiotensin system blockade? It is generally agreed that the AT1 receptor is responsible for the growth-promoting effects of Ang II, but the actions of the AT2 receptor are less well known. We and others have shown recently that the AT2 receptor may inhibit growth19 by antagonizing the effects of the AT1 receptor.20 Despite these observations, the exact physiological function of the AT2 receptor is still not understood. Since AT1 receptor blockade results in an increase in circulating Ang II levels, a shunting of Ang II to the AT2 receptor must be considered. However, this effect would be observed only if the AT2 receptor is expressed in the injured vessel. Pertinent to this, we have shown that injury results in the upregulation of AT2 receptors in the rat carotid artery.19 However, this point is controversial.21 In this context, the article in this issue of Circulation did not address whether AT2 receptors are expressed in the injured porcine coronary artery.15A Furthermore, it is unclear whether the AT2 receptor ligand and the balanced AT1/AT2 receptor ligand are AT2 receptor agonists or antagonists. With ligands of the AT1 receptor, such as the L-158,809 used in this study, the designation as an agonist, partial agonist, or antagonist is relatively clear. However, with AT2-specific ligands or with the balanced AT1/AT2 ligand, the designation of the actions at the AT2 receptor is not straightforward. Recent work in our laboratory as well as work by several other investigators has begun to elucidate the intracellular signaling pathways of this receptor. In most cases examined, it is now generally accepted that the two common AT2 receptor ligands PD123319 and CGP42112 differ in that the former is an antagonist and the latter is an agonist.22 Unfortunately, the designation for the AT2-specific ligand or the balanced AT1/AT2 ligand used in the Huckle et al study15A is not known. Thus, it is not possible to determine, on the basis of this study, the role of the AT2 receptor in neointimal hyperplasia.
Appropriate study design, including an evaluation of the pharmacokinetics, is essential in determining the outcome of any experimental or therapeutic study. A good example is the use of ACE inhibitors to block neointimal development. In the initial study by Powell and colleagues,7 the greatest protection was observed when the drug was administered 1 week before the injury, with diminishing protection when the drug was initiated at the time of injury or after the injury. Further, we demonstrated that the dose of drug necessary for vascular protection was considerably greater than the dose required to elicit a hemodynamic effect.16 Unfortunately, the first clinical studies on the use of the ACE inhibitors to block restenosis used a protocol in which drug was administered after angioplasty and at doses used for blood pressure regulation.14 This negative clinical study must be viewed in light of the requirements dictated by the animal studies. As pointed out by Huckle et al,15A the current generation of ACE inhibitors may not have the appropriate characteristics to allow their use at the levels seen to be necessary in the animal studies.
Was the study designed by Huckle and colleagues15A appropriate? These authors spent considerable effort to demonstrate activity of the AT1 receptor antagonists toward the porcine receptor and to demonstrate that the plasma levels of antagonist were appropriate to block the hemodynamic effects of an Ang II challenge. However, it is not clear whether the dose to block the hemodynamic actions of Ang II is the same as the dose to inhibit the in vivo vascular growth response to Ang II. Even if we disregard the potential contribution of renal, adrenal, and neuronal Ang II receptors, which may influence blood pressure and which may be modified by Ang II receptor blockade, one may still question whether the same drug concentration is achieved in the vessel being injured (large-caliber artery) as is achieved in the vascular beds that regulate the hemodynamic responses to Ang II (resistance arterioles). Thus, to definitively demonstrate appropriate receptor blockade in the injured vessel, an evaluation of hemodynamic response may not be sufficient. A more direct examination of receptor occupancy in the vessel of interest, such as in situ radioligand binding assays, may be required. Similarly, it is difficult to define a dose of the AT2 ligand that would result in adequate receptor blockade. In this article, the authors show that the chosen dose of the AT2-specific ligand does not affect blood pressure, demonstrating lack of crossover to the AT1 receptor. However, sufficient blockade of the AT2 receptor was not demonstrated.
In light of the differences observed among animal models not only with the antagonists of the renin-angiotensin system but with other agents as well, what is the appropriate model for evaluation of agents directed toward restenosis? Experimental models of restenosis differ not only in the animal species but also in the type of artery studied (ie, elastic versus muscular) and in the method of vascular injury. The injury models range from simple endothelial denudation to balloon dilatation with disruption of internal elastic lamina and overstretched medial damage and to frank medial and adventitial dissection with or without stent placement. The severity of vascular cell damage influences the release and expression of growth factors; the magnitude of platelet activation and tissue factor expression determines the degree of thrombus formation; and the extent of adventitial injury and inflammation may affect the amount of adventitial fibrosis and vascular remodeling. As stated by Huckle et al,15A “distinct injuries … may provoke patterns of response that differ in their sensitivity to specific inhibitors.” The range of patterns of response also underscores the potential complexity of tissue injury in human interventional therapy. A further complication is encountered in humans: the human arteries that are subject to interventional treatment are not normal, as in the animal models, but rather are atherosclerotic. Therefore, the contribution of an increasing number of cell types is likely to be seen. For example, in the balloon-injured rat carotid artery, the contribution of macrophages and T cells to the overall proliferative response is likely to be small, whereas in a more complex model or in humans, these cells are likely to contribute significantly to the response. Even the quality and quantity of native disease vary from a fatty lesion to an ulcerated plaque with platelet thrombus to a calcified fibrotic lesion. The difference in the “substrate” of the blood vessel may influence the biological response to angioplasty injury. So far, no animal model has been able to mimic the complexity and heterogeneity of human atherosclerotic lesions.
It is our opinion that strategies aimed at blocking selective biological mediators are limited by the multiplicity of the mediators, the plurality of the cell surface receptors, and intracellular signaling mechanisms. We proposed previously that a successful strategy to inhibit cell proliferation would involve blocking the final common pathway of cell cycle–regulatory genes. Indeed, we and others have shown that antisense oligonucleotide–blocking c-myb, c-myc, cyclins, cd kinases, and proliferating cell nuclear antigen are effective in preventing neointimal hyperplasia in several species.23 24 25 26 Similarly, the use of a transcriptional factor decoy against E2F inhibits the coordinated activation of cell cycle–regulatory genes and consequently neointimal development.26 Moreover, Chang et al27 demonstrated that the transfer of a constitutively active retinoblastoma gene can yield similar positive results in a model of balloon-injured porcine iliac artery. Recently, rapamycin has been shown to be effective in preventing neointimal hyperplasia, and this drug appears to exert its action in part by cell cycle inhibition.28 Since the mechanism of cell cycle progression appears to be conserved in all species, this strategy has been shown to be effective in all species studied thus far. Taken together, these data support our hypothesis that cell cycle inhibition is an effective method of limiting smooth muscle proliferation in vivo. Clearly, the ultimate proof awaits a clinical trial in humans. This strategy is likely to be effective in human disease if smooth muscle proliferation is an important contributor to restenosis.
Given the above discussion, what is the potential use of antagonists to the renin-angiotensin system as therapy for restenosis after vascular intervention? Because of the caveats concerning ACE inhibitor dosage in past clinical trials, we cannot conclude that inhibition of ACE would not prevent restenosis if dosages comparable to those used in the animal studies could be employed. With the characteristics of the current generation of drugs, this may not be possible. Future drug development may result in an inhibitor that may be more tissue-penetrant, allowing the inhibition of tissue ACE without resorting to high levels of drug. However, such an ACE inhibitor would not block Ang II generation by non-ACE pathways. Is there any potential use for the Ang II receptor antagonists? The study by Huckle and colleagues15A is not encouraging; however, sufficient caveats exist that complicate the interpretation. Moreover, uncertainty exists concerning the applicability of the model to human disease. As stated in their article, “our results do not exclude a possible role for Ang II in the vascular response to angioplasty in humans.” Nevertheless, one may conclude, on the basis of these studies, that the renin-angiotensin system does not appear to be a pivotal player in the complex process of restenosis.
Despite the conclusion that the renin-angiotensin system may not be a major contributor to the pathophysiology of restenosis, we caution the reader not to assume that this system is without effect in other physiological or pathophysiological circumstances involving vascular remodeling. An extensive review of this area is beyond the scope of this editorial; however, several lines of evidence from both experimental animal models and clinical trials have suggested that this system may play an important role in angiogenesis, embryonic and fetal development of the vasculature, hypertension-induced remodeling, cardiac hypertrophy, and atherosclerotic lesion formation. Indeed, several clinical trials on the use of ACE inhibitors in atherosclerosis are currently under way, with results due to be released in the near future. Perhaps it will be shown that, unlike the acute structural changes that occur during restenosis, the more chronic changes during atherosclerosis may be more dependent on the effects of this system.
As discussed earlier, Ang II is only one among a myriad of biologically active mediators, such as platelet-derived growth factor, fibroblastic growth factor, transforming growth factor-β, insulin-like growth factor-1, and thrombin, that are activated after vascular injury. Like the renin-angiotensin system, the degree of activation of these factors and their role in vivo appear to differ among species. Indeed, recently angiopeptin, a somatostatin analogue, has been shown to dramatically reduce neointima formation in several animal models, including the porcine coronary artery,29 yet it had no effect on restenosis in a human clinical trial.30 Despite the apparent disappointments with the studies of the inhibition of the renin-angiotensin system, lessons can be learned with regard to studies of these other mediators. Careful studies addressing the points raised in the Table⇑ will help in the evaluation of therapeutic agents directed toward these and other potential mediators of restenosis. However, even if one were able to address satisfactorily the problems summarized in the Table⇑ and discover an agent that is efficacious in multiple nonhuman species and models, one cannot be absolutely certain that the drug would be efficacious in human restenosis. In the absence of a positive clinical trial, one cannot be confident that any of the experimental models are better in simulating human restenosis and in predicting human therapeutic outcome.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|SMC||=||smooth muscle cell|
|VSMC||=||vascular smooth muscle cell|
The opinions expressed in this editorial are not necessarily those of the editor or of the American Heart Association.
- Copyright © 1996 by American Heart Association
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