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Cardiovascular Research 2003 60(2):220-222; doi:10.1016/S0008-6363(03)00542-X
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

New kits on the blot—can we microarray the future of atherosclerosis?

Joerg Herrmann*

Department of Internal Medicine, Mayo Clinic Rochester, 200 First Street S.W., Rochester, MN 55905, USA

*Tel.: +1-507-255-1296; fax: +1-507-255-1824. Email address: herrmann.joerg{at}mayo.edu

Received 30 July 2003; accepted 31 July 2003

See article by Martinet et al. [19] in this issue.

Cardiovascular diseases (CVDs) have a long track record, reaching pandemic proportions with the industrialization of the world. Current estimates of the World Health Organization project that, in less than 7 years from now, CVD will become the leading cause of death in developing countries and that, in less than 13 years from now, 25 million people will die from CVD on a global scale each year [1]. A major proportion of these CVD deaths will be related to atherosclerotic CVD (ASCVD) and, particularly, to its complication stage [2]. Hence, there is a strong call to the cardiovascular research community to identify pivotal mediators of atherosclerotic lesion formation and complication which at some point may turn into therapeutic targets.

What may be considered as searching for a needle in a haystack has been tremendously facilitated by the availability of the microarray technique. This technique, which allows the detection of labeled RNA hybridizing to DNA molecules attached to a solid surface, evolved in a number of steps in the 1960s and 1970s and was finally moved to high capacity in the 1990s [3,4]. Microarray plates with a density of >250,000 oligonucleotides or >10,000 cDNA per cm2 currently serve as a versatile screening probe in genomics research, and it will not be long until arrays become available that allow the screening of the entire genome on one chip for less than one cell and 1 µg of tissue [5,6]. With regard to cardiovascular medicine, DNA microarray assays have already identified a three-digit number of genes potentially involved in atherosclerosis [7–16].

As marvelous as these assays are, they are not without concern. Indeed, a number of potential pitfalls to these kinds of genomic studies have been reported, including array-probe and sample sensitivities and comparability among different assays [17]. However, what seems to be the greatest concern of all is that, for many aspects of cell biology and physiology, the most important circuitry occurs at the protein and not at the mRNA level; this was mentioned by Eric Lander in 1996, who, at that time, already asked for a "chip" detector for proteins [18]. Within five years from this statement such a device has become commercially available, and it is thrilling to read about its first-time use in atherosclerosis research by Martinet et al. in the current issue of Cardiovascular Research [19].

Using this novel, high-throughput immunoblotting technique, Martinet et al. were able to analyze the expression of more than 800 proteins in pooled samples from 12 carotid endartherectomy specimens and seven segments of non-atherosclerotic mammary arteries simultaneously. They identified 15 proteins to be expressed at least five times more or five times less in atherosclerotic tissue compared with non-atherosclerotic tissue; the differential expression being confirmed by standard Western blotting for seven of these 15 proteins. Proteins confirmed to be expressed more included ApoB100 and ApoE, MnSOD, PTP1C/SHP-1, and TSP-2, primarily related to lipoprotein metabolism, oxidative stress, lymphocyte receptor signaling, and angiogenesis; those confirmed to be expressed less comprised GSK-3beta and ALG-2, primarily related to intracellular signaling and apoptosis.

Is this the result we were expecting? In view of the volume of genes projected to be differentially expressed in atherosclerosis by DNA microarray studies, the proteins eventually found to be differentially expressed in atherosclerotic tissue with the use of novel, large-scale immunoblotting seem to differ in quantity and quality. The fact that differential expression was confirmed for only slightly less than 50% of the proteins identified by protein microarray does not indicate that the relatively low yield was a matter of low sensitivity. In fact, the choice of a threshold value of at least five times difference indicates that the assay would be very sensitive in noticing sample differences if allowed to be. Nevertheless, the positive predictive value seems to be relatively low, and there is a strong need to confirm data obtained by the novel protein chip technique with traditional Western blotting. There also seems to be a strong need to carefully review the target composition of a protein microarray; even in plain numbers the current generation of protein chips has a 20-fold lower probe density than the current generation of DNA microarrays. As long as there is uncertainty about their definite target compositions, there will be uncertainty about the true extent of congruency and discrepancy between genomic and proteomic microarray analyses. Nevertheless, with this premise in mind, the first-time use of large-scale immunoblotting in atherosclerosis research brings attention to proteins that were hardly ever related to atherosclerosis before. It is highly commendable that Martinet et al. [19] did not leave it to existing knowledge to give meaning to these new findings, and given their track record in the field, it is highly understandable that their further exploration of the matter would focus on ALG-2 with quite remarkable findings.

In this regard, using real-time RT-PCR, Martinet et al. found that the level of ALG-2 mRNA was not reduced in atherosclerotic plaques, pointing towards the fact that the reduced expression of ALG-2 was the consequence of post-transcriptional modification rather than reduced ALG-2 transcription and highlighting the potential and significance of combined and comparable genomic and proteomic approaches. Using microdissecting techniques, they were further able to demonstrate that a post-transcriptional reduction of intracellular ALG-2 levels was a characteristic of plaque cells but not of normal media smooth muscles cells. Furthermore, in isolated cell culture experiments they found a reduction of intracellular ALG-2 levels in the presence of unaltered ALG-2 mRNA levels once THP-1 macrophages turned into foam cells upon exposure to aggregated or oxidized LDL. Finally, by introducing ALG-2-specific small-interfering RNA into THP-1 macrophages, they were able to demonstrate that reduction in intracellular ALG-2 levels renders these cells more resistant to apoptotic but not to necrotic cell death.

Indeed, overcoming apoptosis is a mechanism with important implications for atherosclerosis. Very recently the observation was made that macrophages can "survive" in atherosclerotic lesions although the caspase-3 apoptotic cascade is triggered within them [20]. The current finding of post-transcriptional downregulation of ALG-2 could be a valuable extension of these previous findings, although the entire subject is certainly more complex. Anti-apoptotic mechanisms have also been ascribed to foam cell formation [21]. However, the fact that reduction of intracellular ALG-2 levels follows rather than precedes foam cell formation makes ALG-2 a less likely mediator in this process. Nevertheless, ALG-2 may be involved in a novel, anti-apoptotic mechanism of foam cells, allowing them to survive in an "unpleasant" environment, albeit not keeping them from undergoing necrotic cell death, which has been associated with lipid pool formation. In as much as macrophages and foam cells seem to be affected by ALG-2, there seems to be little evidence that the same applies to smooth muscle cells, which would be a crucial determinant, for instance, for plaque stability. Previously, Mark Kockx made the valid statement that apoptosis of macrophages could be beneficial but apoptosis of smooth muscle cells detrimental for plaque stability [22]. In view of this statement and the current findings, the question arises as to whether ALG-2 can be viewed as a new detrimental factor in ASCVD.

Obviously, more work is needed to answer this question sufficiently. For instance, it would be interesting to have a more detailed picture of how ALG-2 expression correlates with plaque morphology and the different stages of plaque development. After all, atherosclerosis is a rather dynamic and multifocal process and appropriate attention has to be given to these disease attributes in all their heterogeneity [23]. In addition to parallel approaches such as the current one, future temporal and spatial approaches may also apply protein microarrays which are more extensive in quantity and quality than the current generation [24]. Organization and integration of the volume of information we can anticipate coming in the years ahead seems to be pivotal in line with the Human Protein Project [25]. This will determine the final impact of microarray analysis, and it would be tremendous if this could be marked as beneficial as it has been for DNA microarray in cancer classification and prognostic evaluation [26]. With all the progress made on tissue level, it is still worth remembering that biopsy is not an integral part of the prognostic evaluation of the atherosclerotic plaque, pointing to the need of systemic "biomarkers". Yet, protein microarrays may help to identify new proteins that are specifically expressed in atherosclerotic plaques, particularly in those prone to rupture, and have parallel reflections in serum concentration. In this sense, protein microarrays could contribute to success stories as they have been seen, for instance, for cardiac troponins.

The new kit on the blot clearly shows us for the first time both the opportunities and limitations of high-throughput immunoblotting for cardiovascular research. As for now, we may not to be able to microarray the future of atherosclerosis but we may be able to add microparticles to the array of understanding of this disease process to have the chance to lessen its detrimental effect on the individual and society at some point in the future.


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