Expression Profiling Identifies Smooth Muscle Cell Diversity Within Human Intima and Plaque Fibrous Cap

Abstract

Background-The fibrous cap of the atherosclerotic lesion is believed to be critical to stability because disruption of the cap is the final event leading to plaque rupture. We have, therefore, used expression arrays to define the phenotype of the cap and other plaque components. Methods and Results-To identify unique expression programs able to distinguish the smooth muscle of the cap from other plaque smooth muscle cells, RNA profiles were determined in human carotid artery media, nonatherosclerotic adjacent intima, fibrous cap of advanced atherosclerotic plaques, and whole advanced plaque with cDNA arrays covering 21 000 or 26 000 Unigene clusters. The molecular signature of each tissue was dominated by a core gene-set with differential expression of Ͻ1% of clusters assayed. Conclusions-Both intima and cap expressed novel genes not previously associated with SMC pathology. If the cap is derived from a unique subpopulation, this pattern is the signature of that particular set of cells. Key Words: atherosclerosis Ⅲ cardiovascular diseases Ⅲ genes Ⅲ molecular biology Ⅲ plaque M ost discussions of clonal origins of the atherosclerotic plaque have neglected the obvious question: "Is the smooth muscle cell (SMC) clone a distinct cell type?" 1 ; for example, Williams and Tabas suggested that the matrix produced by intimal SMCs at preatherosclerotic sites may lay down proteoglycans able to bind LDL. 2 Others have suggested that plaque rupture might result from the high apoptotic rate or low replicative life spans of cap cells based on properties seen in cultured plaque SMCs. [3][4][5] Unfortunately, these properties cannot be confirmed in vivo. One way of analyzing differences between tissues is to perform transcript profiling using cDNA arrays with message derived from tissues of interest. We and others have done this to compare distinct vascular tissues in vivo. 6 -9 Many studies have been performed to examine differences in vivo between cancerous and normal tissues. 10 -12 To address this current issue of plaque SMC-specific phenotype, we have performed a largescale profiling of gene expression in normal and atherosclerotic arteries using human cDNA arrays of Ϸ21 000 or 26 000 Unigene clusters. Expression in the fibrous cap was contrasted with expression in normal carotid media, adjacent nonatherosclerotic intima, and the more complicated cellular milieu of the whole atherosclerotic plaque. Methods Isolation and Processing of Tissue SamplesCarotid plaques were removed by endarterectomy, separated into fibrous cap and adjacent nonatherosclerotic intima, or reserved whole. Media were obtained from transplant donors. To obtain sufficient RNA for arraying, cap and intima were distributed into pools. Please see http://atvb.ahajournals.org for detailed Methods and Table I. RNA Purification, Array Hybridization, and Analysis RNA was purified, P 33 -labeled cDNA probes were created from 2 to 4 micrograms of total RNA from each pool per sample, equivalent cpm probes were hybridized with GF200, GF201, GF202, GF203, and GF...

Genome-Wide Expression Studies of Atherosclerosis

“…10,16,27 A cluster of stress-responding genes was shown to be upregulated in the fibrous cap. Adams et al 26 showed more consistent differences in gene expression between the intimal and medial SMC than between the cap and the media, because the adjacent nonatherosclerotic intima appears much more media-like than does the cap.…”

“…18,28 Consequently, this lack of transparency in the morphology of analyzed samples hinders extrapolation and comparison of gene expression results. In only a minority of studies, 10,[25][26][27][28] the expression profiles of samples with a uniform cell content have been compared. However, one of these studies compares the transcriptome of SMC-enriched and macrophage-enriched cell populations 28 and does not exploit the advantage of isolating relatively pure cell populations.…”

“…To this end, 5 recent studies have used permutation-based methods: significance analysis of microarrays 37 (SAM), 13,21 custom-made analysis algorithms including permutations to study multiple sets of time-course microarray data, 9,13 or by comparison with randomized data sets to quantify false-positive frequency in the data sets. 26 The statistical techniques discussed above aim to identify genes that are differentially expressed on a gene-by-gene basis. An alternative method of analyzing microarray data sets is to exploit the correlation in expression patterns between genes that perform similar functions or belong to the same biological pathway.…”

“…To compare gene expression patterns from different parts of the atherosclerotic vessel wall, laser capture microdissection 10,28 and macrodissection 26,27 have been used to isolate relatively pure cell populations from whole mount specimen (Table 3). As mentioned, these techniques help to overcome one of the shortcomings of the whole mount approach as it corrects for the cellular composition of a sample.…”

“…In 3 studies, 7,26,27 macrodissection was used to separate the smooth muscle cell (SMC)-rich fibrous cap, 26,27 media, 7,26 and nonatherosclerotic intima. 26 To this end, the adventitia was trimmed, the endothelium was scraped off, and the fibrous cap was dissected from the necrotic core and shoulder regions. In 2 studies, 10,28 laser capture microdissection was used to dissect SMCs or macrophages from whole mount specimens for subsequent cell-specific RNA isolation.…”