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© 2001 American Heart Association, Inc.
Editorial |
Molecular Markers, Fibrous Cap Rupture, and the Vulnerable Plaque
New Experimental Opportunities
From the Department of Pathology, University of Washington, Seattle, Wash.
Correspondence to Stephen M. Schwartz, Department of Pathology, University of Washington, Box 35-7335, 1959 NE Pacific St, Seattle, WA 98195-7335. E-mail tamid{at}u.washington.edu
Key Words: KW arrays • PCR select • MRI • atherosclerosis • plaque rupture
The study by Faber et al1 in this issue of Circulation Research uses subtraction suppression hybridization (SSH) to identify genes specific to the ruptured atherosclerotic plaque. This article may represent the early days in the oncoming studies of advanced atherosclerosis, a surprisingly neglected area of study.
Part of the reason for this neglect has been the confusion in the past several years over the term "vulnerable plaques"—lesions of atherosclerosis that are thought to be associated with a higher risk for thromboembolic complications. The American Heart Association (AHA), unfortunately, has encouraged the application of this term to a specific morphology.2 The problem is that the AHA classifications scheme implies that we can define critical early and "vulnerable" lesions based on morphology observed at autopsy. Virmani et al3 have offered a simpler, less dogmatic scheme that uses morphological terms without implying mechanism. As that article discusses, a purely descriptive, nonjudgmental approach leads to more focused questions about the specific processes and related morphologies that could be hypotheses for further study. Testing those hypotheses, as we will discuss, may now be possible depending on very new methods that permit noninvasive imaging of the evolving lesions.
Faber et al,1 although using the AHA scheme, do a very good job of specifying the morphology of their lesions. In essence, the ruptured lesions they describe have thin fibrous caps, multiple areas of disruption of that cap, and evidence of acute rupture leading to thrombosis. Lesions with similar characteristics, lacking frank rupture, are the sort called types V and VI by the AHA criteria and asserted to be "vulnerable".2 Unfortunately, autopsy data cannot determine whether such lesions are prodromal, causative, or merely coincidentally associated with the clinical events leading to death from coronary artery occlusion. Arbustini et al,4 for example, found a high incidence of such lesions even in patients with advanced atherosclerosis dying of unrelated causes. Moreover, at least a portion of patients, especially among younger women, appear to die of occlusive thrombosis without any of the stigmata of the vulnerable lesion.5 Risk factors such as smoking, lipoprotein abnormalities, mutations in platelet adhesion molecules, fibrinogen levels, and even hypertension might reflect systemic alterations in the mechanisms underlying thrombosis and coagulation rather than alterations in the probability that lesions will progress to the point of rupture. Indeed, until recently,6,7 we have not been able to determine whether these factors accelerate formation of vulnerable lesions because we have not had animal models that proceed to lesions resembling the AHA’s type V and type VI lesions. Thus, even if present efforts at treating chlamydial infections8–10 are successful in decreasing the incidence of death from atherosclerosis, it will be difficult to tell whether this is because of a change in the rate of plaque progression or whether it is because of alterations in systemic factors such as the probability of coagulation.
While we now have animal models for plaque rupture,6 these models would only have limited value without better knowledge of the process in humans. The usual animal models have not produced advanced lesions, probably because the studies were not carried on for a sufficient period of time. This has recently changed. In contrast, Prescott et al11 studied older, hyperlipemic swine while our group and another group studied older, hyperlipemic mice.6,7 Both species showed advanced lesions, with rupture at specific sites of the vasculature. The swine model, unfortunately, is of limited use because advanced, ruptured lesions have only been demonstrated in experiments that begin with juvenile animals. These animals grow to become full-sized pigs too large for most types of experimental study. Duplication of this work in smaller pigs is very desirable.
The murine experimental lesions6,7 are probably more useful because of the size of the animals, short life span, and low cost. Nonetheless, we need to express concern that critical factors leading to plaque rupture in minute murine vessels may be different from the critical factors in human disease. Obviously, knowledge of the process in humans is critical.
The ultimate test of any hypothesis about plaque rupture7,11 will depend on technologies that would allow us to serially image advanced lesions by noninvasive studies in humans. The best chance for such imaging comes from recent developments in MRI of the advanced atherosclerotic lesion in the carotid artery and aorta. The Figure shows the correlation between magnetic resonance images and histological examination of carotid endarterectomy specimens. Studies from our group12 have demonstrated a high level of agreement between in vivo MRI and histological examination of this tissue for the identification of fibrous cap characteristics. Soila et al13 and Maynor et al14 published early reports demonstrating that lipid components of atherosclerotic plaque could be distinguished with MRI. Toussaint et al15 showed that MRI examination of this tissue was useful for the identification of fibrous cap characteristics. They found that calcification, fibrous intimal tissue, and hemorrhage could be identified based on T2 measurements of carotid plaques in vivo.15 More recently, Fayad and colleagues16 have demonstrated that MRI is capable of assessing thickness, extent, and composition of atherosclerosis in the thoracic aorta. Additionally, Shinnar et al17 have demonstrated that MRI is capable of identifying carotid plaque constituents with high sensitivity and specificity ex vivo. While these images may still appear difficult to interpret to the reader who is not an expert, improvement in the technology is rapidly occurring and may one day be seen as the key to what we will have learned about the mechanisms leading to atherosclerotic progression.
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Of course, no morphological method can replace the search for specific molecules exemplified by the study by Faber et al.1 In that spirit, the most obvious issue is whether or not the genes found by Faber and colleagues can give us new insights into plaque rupture. Should we expect rupture-specific genes to distinguish between lesions? Surprisingly, Faber et al1 report finding perilipin only in those lesions showing an acute rupture. Perilipin was not found in similar vulnerable lesions with areas of necrotic core that appear no different from the areas of the ruptured lesions showing their molecule. Thus, these investigators may have found a very exciting causative factor for rupture or perhaps merely one of several molecules expressed after rupture occurs.
Perilipin is unlikely to be the sole marker of rupture. Describing one gene out of the 35,000 candidates that may be involved in this process represents at best only an early step. Although the era of plaque imaging may be dawning, the era of being able to discover single or even a few new genes, as represented in the present study, is coming to a close. Current estimates are that the total number of sites of expression of RNA in the genome is approximately 35,000. The technology for representing all of these sequences in a hybridization array is already nearly present in the current generation of microarrays. Thus, in theory, one should be able to take RNA from a ruptured plaque and from a nonruptured plaque and use a large-scale expression array to detail all possible differences in expression. Single observations, like those in the present study, will come to be seen as anecdotal.
If arrays can provide such systematic information, then one needs to ask whether or not other methods, eg, the SSH used by Faber et al,1 are valuable. The answer comes from the technological limits of present technology. First, current arrays do not contain all possible sequences. SSH offers the possibility of finding sequences not on the arrays or, perhaps even more importantly, not present at high enough levels in the sample to be detected in an array. SSH is a derivative of representational distributional analysis (RDA). Chang et al,18 for example, used RDA to detect a rare herpesvirus responsible for Kaposi sarcoma.19 RDA is useful in finding viruses because the method is sensitive to even small sequence differences between two collections of DNA. Adapting this genomic DNA technique to RNA was an obvious step and has been adapted to provide differential analysis of cDNA collections (www.Clontech.com/pcr-select/index.shtml). It is important to realize that SSH is similar to array display, in that both methods, at least in theory, have the capacity of finding all expression differences between two specimens. The first step in SSH is a normalization where the target RNA is hybridized against a large excess of driver. In effect, this should normalize all shared sequences, leaving only the differentially expressed sequences present. This normalization, again in theory, means that SSH is likely to be superior to array display in its ability to detect sequences represented in low abundance, even when all sequences are represented on the array to begin with. In theory, SSH should even be able to find sequences not present on an array.
There is a limitation to SSH imposed by expense. The subtractand, that is, the collection of sequences resulting from hybridization and then differential PCR amplification, is itself a complex mixture that may contain a large number of different cDNAs. The traditional approach, as used by Faber et al,1 is to sequence clones derived from this collection.1 The authors only sequenced a few clones. A larger effort at sequencing would have yielded more differences. Although the total number of distinct clones may be in the tens or hundreds, the cost of sequencing a large number of clones to identify the unique clones is prohibitive. An alternative, not used in the present study, would be to hybridize the subtractand against a large array. This should detect differentially expressed sequences, although it would not provide any quantitative information and would only be valid to the extent that the sequences are already represented on the array.
In summary, Faber et al1 provide an important transitional study. Clearly, the tools for systematic expression analysis and plaque imaging are getting better and less expensive. Such tools, however, can only be as good as the model systems providing tissue for analysis. New animal models and MRI should offer us much better experimental opportunities to test specific hypotheses about plaque rupture.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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