Antisense Therapy for Angioplasty Restenosis
1995; Lippincott Williams & Wilkins; Volume: 92; Issue: 7 Linguagem: Inglês
10.1161/01.cir.92.7.1981
ISSN1524-4539
AutoresMartin R. Bennett, Stephen M. Schwartz,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoHomeCirculationVol. 92, No. 7Antisense Therapy for Angioplasty Restenosis Free AccessResearch ArticleDownload EPUBAboutView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticleDownload EPUBAntisense Therapy for Angioplasty Restenosis Some Critical Considerations Martin R. Bennett and Stephen M. Schwartz Martin R. BennettMartin R. Bennett From the Department of Pathology, University of Washington, Seattle. and Stephen M. SchwartzStephen M. Schwartz From the Department of Pathology, University of Washington, Seattle. Originally published1 Oct 1995https://doi.org/10.1161/01.CIR.92.7.1981Circulation. 1995;92:1981–1993Percutaneous transluminal angioplasty is now a well-established and frequently performed procedure that has an initial success rate in reestablishing arterial patency of >95%.12 Although good symptomatic improvement occurs in the majority of cases, the procedure is complicated by restenosis in >30% of cases.34 Despite the apparent success of several therapeutic modalities in animal models, attempts to use pharmacological therapy to prevent restenosis in the clinical setting have not been successful. This failure has prompted research into alternative forms of intervention, including the use of antisense oligonucleotides therapeutically targeted to genes believed to be critical for the pathogenesis of restenosis. The rationale for the use of antisense oligonucleotides to prevent restenosis is twofold. First, the prevailing view is that restenosis is the end result of a reactive proliferation of cells of the vessel wall after angioplasty. Thus, it follows that an agent that suppresses cell proliferation may suppress restenosis. Second, antisense agents have been used extensively to analyze genetic events associated with cell proliferation and the cell cycle (review in References 5 and 6). When any cell replicates, there is a characteristic sequential activation of a cascade of genes.78 This cascade of gene activation is also seen as cells are induced to proliferate after arterial injury.9101112 Because antisense agents can suppress the expression of genes associated with cell replication, the use of these agents to block cell proliferation after angioplasty is an attractive concept. Several studies have attested to the efficacy of antisense oligonucleotides directed at cell-cycle proteins in preventing neointimal formation after injury in animal models. The success of these animal studies has spawned widespread interest and enthusiasm for the use of antisense agents to prevent human restenosis. With that in mind, we review the critical issues that may determine whether or not an antisense strategy is likely to be successful in preventing human restenosis. The issues to be considered are as follows. (1) Is replication a critical step in restenosis? (2) Are antisense agents truly specific for their putative targets? (3) What factors determine the specificity and efficacy of an antisense agent? (4) Are side effects likely to be manifested as problems in clinical toxicology? Pathogenesis of Angioplasty Restenosis Before discussing therapy, we need to agree on what we mean by restenosis. When an artery is dilated by angioplasty there is an "initial gain" in lumen size. Restenosis can best be defined as a "loss of gain,"13 that is, a late return of the vessel lumen to a size approaching that upon initial dilatation. It is important to note that this definition describes restenosis in terms of lumen size but does not attempt to identify the mechanisms involved in the changes in lumen size at angioplasty or in restenosis. Indeed, the mechanism involved in increasing the lumen size at angioplasty, the "acute gain," is still not entirely clear. Some of the most illustrative findings on the effects of angioplasty on the vessel wall have come from recent ultrasound studies that suggest that only a small amount of actual plaque mass is lost from the lesion site. Rather, most of the acute gain appears to be due to fracture and compression of the plaque, with fractures of the internal elastic lamina and overstretch of the vessel.141516Next, it is essential to define "success" in the context of restenosis. After angioplasty, about 70% of patients have a persistently dilated vessel that approaches the desired lumen size. We can call this persistent dilatation "success" (Fig 1). In contrast, we can define loss of gain as "failure" and divide it into early and late forms. Early failure occurs when the lumen is occluded by a thrombus or by rapid recoil of the stretched vessel.1317 These processes occur within hours or at most within a few days after angioplasty and are generally considered not to be part of restenosis. However, it is very important to remember that subclinical early recoil, combined with the mechanisms discussed below, may also be an important contributor to what appears to be late loss of gain ("Late Failure A" in Fig 1). The two possible mechanisms for late loss of gain are also shown in Fig 1. The first mechanism to consider is remodeling. Remodeling is a normal process that vessels use to maintain an appropriate lumen size or caliber, particularly in response to changes in blood flow.18 In early atherosclerosis, there is dilatation of the affected vessel.19 This initial dilatation may be analogous to remodeling seen after physiological changes in blood flow. However, when atherosclerosis becomes severe, the lumen size appears to be reset to an inappropriate caliber.19 Therefore, in a real sense, the goal of angioplasty is to prevent the vessel from healing and restoring itself to the inappropriate caliber after dilatation. Nonatherosclerotic vessels can remodel sufficiently to accommodate extensive amounts of intima.19 Thus, we might achieve success despite intimal hyperplasia if the vessel were somehow able to restore itself to a normal caliber by remodeling. Equally, remodeling itself after angioplasty may cause restenosis without increasing vessel wall mass.2021 Unfortunately, virtually nothing is known about the molecular mechanisms involved in remodeling. As a result, to the extent that remodeling is critical to restenosis, we lack clear pharmacological strategies. The second mechanism to consider is neointimal formation. When vessels are injured by any of a variety of processes, they respond by forming a new layer of intima.22 This mass of neointima can narrow the lumen. This would be an especially important mechanism if it were superimposed on an acute but less than critical extent of elastic recoil, as already noted. The extent to which neointimal formation will narrow the lumen is, of course, dependent on remodeling ("Late Failure B" in Fig 1). If remodeling restores the vessel wall to its preangioplasty dimensions, restenosis could occur without any intimal hyperplasia ("Late Failure C" in Fig 1). On the other hand, some degree of intimal hyperplasia may be tolerated if remodeling permits some compensatory dilatation ("Success" in Fig 1). Neointimal formation is the result of cell migration from the intima or media, followed by cell proliferation and connective tissue formation. Although each process may contribute to neointimal mass, cell proliferation is the major source of smooth muscle cell accumulation in the neointima in the most often studied animal model, that of balloon injury to the rat carotid artery.22 Cell proliferation is a dramatic event in the injured rat vessel, in which three generations of replication occur within 2 weeks and can more than double the mass of the vessel.22 At present, however, we do not have evidence for a similarly dramatic proliferative event in the response of the human atherosclerotic vessel to angioplasty. The literature contains two contradictory papers. Strauss et al23 reported extremely high levels of staining for markers of proliferation in atherectomy tissue from both primary and restenotic lesions. However, the values for proliferation in this study are suspect because high values were seen even months after angioplasty and because relatively few specimens were studied. Furthermore, values in primary atherosclerotic lesions were much higher than those described by others using autopsy or surgical excision tissue.2425 In contrast, an extensive study by O'Brien et al26 found only low values of proliferation even early after angioplasty. As pointed out by the latter authors, the possibility remains that replication in the clinical setting is too transient or at too low a level to be detected by random atherectomies; replication might also occur in layers of the vessel wall deeper than are usually sampled. It is also important to realize that the injured wall may produce new extracellular mass via mechanisms that are independent of proliferation. Collagen, elastin, and proteoglycans may all contribute to the loss of gain by forming a mass that occludes the lumen. Finally, a decrease in lumen caliber could occur by retraction or contraction of healing tissue in the wound. This latter mechanism would produce restenosis even if there were no actual increase in tissue mass. In summary, restenosis is defined as a late loss of gain occurring weeks or months after angioplasty. The potential contributing processes include remodeling, healing of the injured vessel, smooth muscle cell proliferation, smooth muscle cell migration, and formation of new extracellular matrix. Any component of these interrelated processes is a theoretical target for intervention, and pharmacological approaches to each have been proposed (reviewed in References 27 and 28). Antisense: A Focus on Proliferation Despite the complex possibilities just discussed, antisense approaches to restenosis have thus far focused only on smooth muscle cell proliferation. The focus of antisense strategies on proliferation reflects several issues. First, as we will discuss below, cell proliferation has several key steps. Blocking any one of these steps is sufficient to halt the entire cascade leading to DNA synthesis. In contrast, the other processes described are either poorly defined at a mechanistic level (remodeling) or lack any single molecular target that could stop the entire process (migration and matrix synthesis). Second, antisense approaches are most effective when the mRNA being targeted is of low abundance (see below). Many mRNAs required for replication are of low abundance and therefore make good targets. Finally, replication may be an early, transient event that follows angioplasty. This proposal is based on the absence of replication in human atherectomy specimens at late time points26 and on results from animal studies using both antisense and conventional drugs in which blockade of replication early in the response to injury had surprisingly long-term effects on neointimal mass, which were seen weeks later. All these factors make replication easier to target than the other, more poorly defined, components of the response. However, it should be borne in mind that if proliferation is only a small part of restenosis after angioplasty in humans, as suggested by some studies,26 then an antisense approach that targets proliferation alone may not work in humans. Principles of the Antisense Approach To understand how antisense agents suppress proliferation in animal models of restenosis or eventually how they may work in humans, it is necessary to analyze how antisense oligonucleotides themselves block gene expression. The synthesis of cellular proteins (whether structural components, enzymes, receptors, or proteins involved in cellular proliferation) occurs via a coordinated sequence of molecular events (Fig 2). The antisense approach to inhibiting gene expression is to block any one of the following processes: uncoiling of DNA, transcription of DNA, export of RNA, DNA splicing, RNA stability, or RNA translation. A large number of antisense approaches have been developed, including the use of antisense oligonucleotides, antisense mRNA (antigenes), and autocatalytic ribozymes (RNA molecules with enzymatic activity) and the insertion of a section of DNA to form a triple helix. The complexities of each approach are beyond the scope of this article (see References 5, 6, and 29 through 31 for reviews); rather, this review focuses on the use of antisense oligodeoxynucleotides, the most common antisense agent in use and the most extensively studied in vascular smooth muscle cells in vitro and in restenosis models in vivo. How Antisense Oligodeoxynucleotides Work Many studies have used the properties of antisense oligonucleotides to inhibit gene expression in cultured cells, and their use has also been extended to whole organisms (reviewed in References 6, 32, and 33). These studies have shown that antisense oligodeoxynucleotides targeted to cellular or viral RNA sequences can reduce target gene mRNA and/or protein product34 and exert biological effects, manifested usually as a suppression of cell proliferation or differentiation. Theoretically, therefore, antisense oligonucleotides are extremely useful agents for targeting any gene sequence in vitro or in vivo. Antisense nucleotides are short (usually <30 bp) complementary DNA or RNA sequences that will hybridize to a specific mRNA forming a hybrid duplex. Although the precise mechanisms by which antisense oligonucleotides reduce target mRNA and protein levels within the cell are imperfectly understood, two main mechanisms have been postulated. First, oligonucleotides have been suggested to exert steric interference to ribosome binding and translation or to splice excision. Evidence for steric interference comes from studies in which antisense to the 5′ cap of the mRNA has been found to be most effective in inhibiting rabbit β-globin protein synthesis (reviewed in Reference 32); the 5′ cap is where a number of initiation factors bind for ribosome assembly, unwinding of DNA, and ribosome translocation along the mRNA.35 Second, it has been postulated that much of the effect of antisense oligonucleotides is due to induction of cleavage of mRNA by the nuclease RNase H, which is widely present in mammalian cells and specifically recognizes DNA-RNA duplexes.36 In the presence of RNase H, oligonucleotides directed to various parts of the coding and upstream sequences of a mouse globin mRNA were shown to be equally effective at inhibiting translation. Furthermore, when the enzyme was blocked by addition of a competitor DNA-RNA hybrid (poly dT/rA), there was a marked reduction in efficacy of globin mRNA degradation.37 Most of the effect of chemically modified oligonucleotides (see below), eg, phosphorothioates, may be explained by this mechanism, and sequences directed downstream of the initiation codon usually fail to inhibit translation unless the hybrid is cleaved by RNase H. Antisense oligonucleotides can also enter the nucleus, where they may inhibit splicing,3839 preventing the processing of pre-mRNA or mRNA, or block transport of the mRNA out of the nucleus. Introduction of antisense oligonucleotides thus results in reduction in specific mRNA and protein levels if mediated by RNase H or reduction in specific protein levels if mediated by steric interference. Advantages of Antisense ApproachesThere are many ways that cell proliferation can be inhibited pharmacologically, so what are the advantages of using antisense approaches compared with conventional inhibitors? The first major advantage of synthetic antisense oligonucleotides, at least in theory, is the potential for design of agents with target specificity. The hybridization of base sequences between nucleic acids is very specific; only the complementary base (C-G, A-T) should be bound. Because the mRNAs of related proteins often have areas lacking significant homology, this specificity of base pairing means that an antisense sequence of bases should target only a single mRNA, without affecting the mRNAs of other genes. As evidence of this specificity, studies have shown that mRNAs can discriminate between oligonucleotides that differ by one or two bases.404142 In the latter two studies, changes in a c-myc antisense sequence of only two bases resulted in almost complete loss of activity. The ability of antisense oligonucleotides to discriminate between mRNA sequences that differ by only a few bases has also been demonstrated and can be used to selectively target the mRNA of a mutated gene. In a study by Saison et al,43 oligodeoxynucleotides directed against a point mutation in the Ha-ras gene could selectively inhibit expression of the mutant gene but not the normal gene. This specificity of binding is greater than can be achieved with most conventional pharmacological inhibitors, which frequently act on a variety of proteins with different binding affinities. However, although the specificity of binding of oligonucleotides is attractive in concept, it is not always achieved in practice (see below). The second advantage is that antisense oligonucleotides targeted to specific mRNAs are much easier to design and synthesize than any previous class of drugs. The structures of oligonucleotides are relatively simple, consisting only of possible polymers of the four base pairs. Since the target sequence is known, rational drug design against a target is theoretically obvious without screening thousands of products as occurs with pharmacological agents. Also, antisense drugs have the potential for permanently altering the target tissue. Constitutive expression of antisense RNA in the target tissue can be achieved by inserting DNA into the host chromosome. In practice, this is usually not done with oligonucleotides but rather with full-length antisense mRNA sequences (antigenes). Finally, if delivered properly, the effects of polynucleotide-based drugs should be highly localized. Nucleotides are taken up into cells and are trapped in the intracellular compartment.44 Any polynucleotide that remains outside the cell or undergoes exocytosis is likely to be rapidly degraded by serum nucleases (see below). This mechanism may help to restrict local delivery of antisense oligonucleotides to the site of delivery. Indeed, site specificity is a very important consideration if one is to target replication in restenosis. Potent antiproliferatives used in cancer chemotherapy almost always have systemic side effects that would be unacceptable in drugs used against restenosis. The side effects of these agents can be reduced by engineering antiproliferative drugs with target site specificity. Such drugs work well for tissues with hormone-sensitive proliferation pathways, such as breast or prostate. However, site-specific inhibitors of proliferation cannot, as yet, be used in the vessel wall because no smooth muscle–specific pathway has been shown to exist. Targets for Antisense Agents Directed Against Proliferation The obvious question arises of what the most appropriate target is for an antisense approach to inhibit replication in the vessel wall. Cell proliferation involves the complex interactions of mitogen binding to receptors, intracellular signal transduction pathways, and changes in the expression of specific genes. Mitogens may affect several intracellular signal transduction pathways, and pathways are branched, connected, interdependent, and in some cases redundant. This redundancy makes it unlikely that blockade of a single receptor or signaling pathway will be sufficient to suppress proliferation. In contrast to the many possible receptors and signal cascades, translation of genes concerned with proliferation is the requisite final common path into which all signal transduction pathways involved in replication converge. A large number of gene products are newly synthesized during the cell cycle and have been shown to be critical to cell-cycle progression (see Reference 45 for review). These gene products include enzymes involved in DNA and nucleotide synthesis (eg, thymidine kinase and DNA polymerases), DNA binding proteins and transcription factors (eg, c-myc, c-fos,c-jun, c-myb), and cell-cycle regulators (eg, cdc-2, cdk-2, and the cyclins). On theoretical grounds, these gene products may be the most effective targets to inhibit proliferation in smooth muscle cells. Since few conventional pharmacological inhibitors of these gene products exist, the use of antisense agents directed against growth-regulatory or cell-cycle genes remains attractive. General Considerations for Use of Antisense Agents Three criteria must be met for antisense agents to be useful experimentally and therapeutically. First, the antisense agent should be stable in vivo, both intracellularly and extracellularly. Second, the antisense agent must be capable of entering cells and binding to the target sequence with relatively high affinity, at concentrations that do not exert significant toxicity to the cell. Third, hybridization to the target sequence should induce suppression of gene expression of the target, and to no other nucleic acid sequences, or to intracellular proteins or lipids. Based on these criteria, a number of physicochemical characteristics of the oligonucleotide are considered when a sequence is selected for use as an antisense agent. In particular, the optimal length, target gene sequence, stability and uptake of the oligonucleotide, and nonspecific effects due to the agent all must be addressed.Length of oligonucleotide. An oligonucleotide used for study should be long enough to be unique to the target mRNA but no so long that it binds to multiple mRNA species nonspecifically. Based on the complexity of the human genome, with approximately 3 to 4 million bases, it has been calculated that the shortest sequence required for recognition of a unique sequence is 12 to 15 bases.46 In practice, most studies have used oligonucleotides of 15 to 30 bases. Increased length of oligonucleotide should improve binding and thus hybrid stability. However, this advantage is offset by an increase in the potential for binding to nontargeted sequences (see below), and longer oligonucleotides may also have variant uptake characteristics. Target sequence. A number of theoretical considerations help in the choice of target sequence for antisense oligonucleotides within a specific mRNA. As most antisense-mRNA interaction is proposed to occur within the cytoplasm, areas of the mRNA with little secondary structure should offer attractive targets. This frequently means sequences directed around the initiation codon of the mRNA. For interactions involving nuclear mRNA, splice sites involved in mRNA processing and export have also been found to be effective. Other sites that have been found to be particularly effective are related to the 5′ cap; the 5′ cap is where a number of initiation factors bind for ribosome assembly, unwinding of DNA, and ribosome translocation along the mRNA (Fig 2).35 Despite these considerations, however, a few base-pair shift in target sequence can profoundly affect the ability of an oligonucleotide to inhibit gene expression. In addition, sequences directed at different parts of the same mRNA have widely differing activities (eg, see References 47 and 48). Although the secondary structure of the mRNA may be partly responsible for differences in hybridization, the full explanation of this phenomenon is unknown. This makes design of oligodeoxynucleotide sequences an informed guess at best, and many sequences are usually tested before sequences are chosen that exert maximal suppression of target gene expression.47Uptake and stability. A further problem of antisense delivery into cells or tissues relates to uptake and stability of sequences. In cell culture, oligonucleotides are usually microinjected into cells or added to the culture medium, whereupon they are taken up into cells. Microinjection is feasible only for small numbers of cells, and therefore most studies in cultured cells use direct addition to the culture medium. However, the exact mechanism of oligonucleotide entry into cells by use of this method is unclear. Oligonucleotides are typically 15 to 30 bp long with molecular weights from 4500 to 9000 D. Oligonucleotides are also polyanions and cannot passively diffuse across cell membranes. Uptake depends on length of oligonucleotide, overall charge and hydrophilicity/lipophilicity (which in turn depend upon chemical modifications of the oligonucleotide; see below), and concentration of oligonucleotide. Uptake is also an energy-requiring process that is maximal at 37°C.49 Studies using fluorescent acridine–labeled oligonucleotides have suggested that uptake of unmodified sequences is by a mechanism consistent with receptor-mediated endocytosis, and two surface proteins (34 and 80 kD) have been identified that may mediate the process.5051 However, this route of uptake has yet to be conclusively proven, and it is also likely that the predominant method of uptake differs among modified oligonucleotides.49 For instance, it has been shown that the 80-kD protein binds phosphodiester and phosphorothioate oligonucleotides but not methylphosphonates.5052 Movement of oligonucleotides across cell membranes is also not a one-way process. Oligonucleotide exocytosis has been demonstrated in a number of cell types,49 being temperature dependent, maximal at 37°C. Uptake of both phosphodiesters and modified oligonucleotides is generally an inefficient process,53 but it can be enhanced by complexing the oligonucleotide sequence with liposomes5455565758 and/or by using a virus transport system such as that used by the hemagglutinating virus of Japan (HVJ).59 Use of liposomes masks the negative charge present on many types of oligonucleotides, particularly unmodified and phosphorothioate-modified sequences, and may thus allow diffusion across the cell membrane. The HVJ-liposome system also bypasses receptor-mediated endocytosis; the HVJ-liposome complex fuses directly to the plasma membrane at neutral pH and can release DNA contained in the core of the complex into the cell. Addition of a nonhistone nuclear protein to the complex apparently results in translocation of the DNA sequence to the nucleus. DNA delivered in this way apparently shows a 10-fold higher incorporation into cells in culture than DNA-liposomes alone.60Although evidence indicating that antisense oligonucleotides suppress target mRNA levels suggests that oligonucleotides do enter the cell, direct evidence of cellular uptake comes from studies in which antisense oligonucleotide–RNA duplexes were directly demonstrated within the cell by S1 nuclease analysis.394161 Many studies have also used radiolabeled or fluorescence-labeled oligonucleotides to monitor uptake and distribution of oligonucleotide within cells (Fig 3). In most cells, uptake is first demonstrated in a granular pattern consistent with internalization into endocytotic vesicles.57 At later time points, and particularly if complexed with liposomes, nuclear staining of antisense sequences becomes evident, indicating transport to the nucleus.57 Although in general uptake of oligonucleotides is poor, a recent study in human vascular smooth muscle cells demonstrated oligonucleotide uptake at 1 hour with persistence of full-length oligonucleotides within cells up to 16 hours.62 Thus, although the precise mechanism of entry has not been ascertained, the fact that the sequences do enter the cell has been established. Delivery of antisense oligonucleotides in vivo to the arterial wall has been achieved by two methods: direct transfection and HVJ-liposome–mediated uptake. For example, a single application of phosphorothioate-modified oligonucleotides in a gel matrix to the adventitial surface of a rat carotid artery after injury can suppress target mRNA levels.424863 However, intraluminal instillation with interruption of blood flow also appears to be effective in both the rat carotid artery and the pig coronary artery,596064 and intravascular delivery can be enhanced by complexing the oligonucleotide with HVJ-liposome. With this latter system, significant uptake of oligonucleotide in the arterial wall can be observed after only 10 minutes, and oligonucleotides show persistence in the arterial wall up to 2 weeks after administration.60In addition to generally poor uptake, the instability of oligonucleotides has been a significant problem in their use in vitro and their potential use in vivo. Oligonucleotides are very sensitive to degradation by exogenous and endogenous nucleases (phosphodiesterases).65 These enzymes are widespread, with significant activities being demonstrable in serum,6667 and the presence of nucleases has previously precluded the use of unmodified oligonucleotides in studies of whole cells (but not all studies; see Reference 41). To improve stability against nucleolytic phosphodiesterases, the phosphate backbone of the oligonucleotide has been chemically modified in a variety of different forms (Fig 4).6768 Compared with the unmodified phosphodiester linkage, chemical modifications such as phosphorothioate and phosphoroamidate bonding have improved nuclease resistance by up to 10-fold, thereby reducing the concentration at which a biological effect can be observed.6970 These modifications, particularly the methylphosphonate form, can also increase cellular uptake significantly by removal of the net negative charge from the compound. Despite some reduction in the ability of modified agents to hybridize to the target sequence7172 and increased nonselective inhibition of translation,73 modified oligonucleotides in general and phosphorothioates in particular are widely considered to be the most promising agents for therapeutic use.74Despite the use of modified oligonucleotides, the inhibition of gene expression using antisense
Referência(s)