Published online before print November 17, 2005, doi: 10.1161/01.ATV.0000196647.45718.d6
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© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Expression Profiling Identifies Smooth Muscle Cell Diversity Within Human Intima and Plaque Fibrous Cap
Loss of RGS5 Distinguishes the Cap
From the Department of Pathology (L.D.A., S.M.M.), Center for Cardiovascular Biology and Regenerative Medicine, and the Department of Biostatistics (A.R.), University of Washington School of Medicine, Seattle; and the Division of Surgical Sciences (R.L.G., J.L.), Wake Forest University School of Medicine, Winston-Salem, NC.
Correspondence to Lawrence Adams, University of Washington, Department of Pathology, 815 Mercer St # 419, Seattle, WA 98109-4714. E-mail ladams{at}u.washington.edu
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Abstract |
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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 <1% 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. The loss of RGS5 in the fibrous cap is of particular interest because of its role in vessel development and physiology.
Expression profiles of whole plaque and plaque subcomponents (media; adjacent nonatherosclerotic intima; and fibrous cap) were obtained on filter sets containing 21 000 or 26 000 Unigene clusters. Unique patters of gene expression characterized each layer. One remarkable difference was the loss of RGS5 expression in the fibrous cap compared with adjacent nonatherosclerotic intima and media.
Key Words: atherosclerosis • cardiovascular diseases • genes • molecular biology • plaque
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Introduction |
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Most 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.3–5 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 large-scale profiling of gene expression in normal and atherosclerotic arteries using human cDNA arrays of
21 000 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 |
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Isolation and Processing of Tissue Samples
Carotid 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, P33-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 GF204 cDNA arrays (GF204, plaque and media only; Research Genetics), and filters were analyzed as previously described.6 Each filter represents 5000 unique clusters. Data were normalized and analyzed using Pathways (Research Genetics). Genes were defined as expressed in a particular tissue if labeling intensity was >2-fold the average background measurement on each filter for all samples of that tissue type.
Tissue Histology, Immunohistochemistry, and In Situ Hybridization
These all used standard methods.6,7 Please see http://atvb.ahajournals.org for details. Array data were validated by immunostaining for NMMHC-B (1:500, a kind gift of Dr Nagai, University of Tokyo Department of Cardiovascular Medicine); 1:100, connective tissue growth factor (R&D Systems); versican (Developmental Studies Hybridoma Bank, University of Iowa); and by in situ hybridization for RGS5 using a human cDNA as described previously.6,7
Statistical Methods
Data analysis emphasized consistency of results; all genes declared differential in a tissue (numerator tissue) were always expressed higher in that tissue than the denominator tissue. We performed a multiple unpaired sample analysis using the ratios between all possible combinations of pairs of array probes for any two tissue types being compared (keeping the proper numerator/denominator relationship) following the Ly et al unrelated pair-wise comparison methodology for array data analysis.13 This allows us to include all possible sources of patient variation which might result from the unpaired nature of the data.
Genes were defined as "differentially expressed" between any two tissue-types if all combinations of sample pair ratios were >1.3-fold in the same direction (tissue) and the average of these ratios (the mean combinatorial ratio, defined as the average value over the set of ratio combinations in the multiple unpaired sample analysis) was >1.5-fold with a standard error of <2/3 the mean combinatorial ratio. These cutoff values have been previously described.6,7,14 Standard errors were calculated from the ratio values in the multiple unpaired analyses.
To quantify false-positive frequency in the data sets, we flipped the relative expression for half of each data set, producing 3 unique randomized data sets (6 sets, 3 of these being mirror images of the other 3 and producing the same ending data) for each two tissue comparisons, as we have previously described.7 For example, for the four intima (I) samples I1 to I4 over media (M) (M1–3 is shorthand for all 3 medias; ie, I1/M1–3 is [I1/M1, I1/M2, I1/M3]), the proper order of numerator to denominators is I1/M1–3, I2/M1–3, I3/M1–3, I4/M1–3. The three random sets constructed to look for spurious positives were: I1/M1–3, M1–3/I2, I3/M1–3, M1–3/I4; I1/M1–3, I2/M1–3, M1–3/I3, M1–3/I4; I1/M1–3, M1–3/I2, M1–3/I3, I4/M1–3. These sets were sorted with the same rules used for the actual data. Data are presented graphically to fit word restrictions. Detailed statistical methods and data tables will be made available on CD on reader request.
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Results |
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Histology of Media, Intima, Fibrous Cap, and Whole Plaques
Cellular complexity is a major concern in any array-based tissue analysis, especially in a complex tissue such as the plaque which contains SMCs, macrophage, lymphocytes, and other inflammatory cells.15–17 To minimize this problem, at the time of surgery, we dissected the carotid lesions into their three components. Areas designated as "fibrous cap" were identified grossly as white, fibrous regions overlying the plaque necrotic core and dissected free of underlying lesion (Figure 1A). Adjacent nonatherosclerotic intima was peeled from the internal elastic lamina at sites lacking gross atherosclerosis. Separation of fibrous cap from plaque was also confirmed histologically. Cap histology usually consisted of a dense fibrous matrix of varying thickness similar to dense connective tissue, sparsely populated by SMCs with thin extensions of cytoplasm (Figure 1B and 1C). In some cases, the SMCs had the "myxoid" appearance seen in restenotic tissue.18 Histological studies of
1/3 of the 40 caps examined showed small numbers of adherent macrophage representing inflammatory attachment to the underlying plaque. The histology of the 40 intimas examined consisted of a loose fibrous matrix populated largely by smooth muscle with abundant cytoplasm. In contrast to the whole plaque, the intima showed only few macrophage or other inflammatory cells (Figure 1D). The whole plaque had the usual complex appearance. To obtain sufficient RNA for profiling, intima or cap was pooled. For details on pooling, average numbers of expressed genes, and other quality control issues, please see Criteria Section I in the online data supplement.
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Intimal Expression Phenotype
Intimal expression of four pools was contrasted with three individual samples of normal carotid media. Consistent differential expression of 48 genes distinguished intima from medial SMCs (0.22% of genes examined; Figure 2A; Table II, available online at http://atvb.ahajournals.org). Only one of these 48 genes, myosin 5C, was more highly expressed in the media. The remaining 47 were intimal. Genes overexpressed on the array in intima and previously associated with human intima, included nonmuscle myosin heavy chain-B (NMMHC-B)19; connective tissue growth factor (CTGF)20; major histocompatibility complex, class II, DR alpha (HLA-DRA)21; and vascular cell adhesion molecule-1 (VCAM-1).22,23 NMMHC-B (Figure 3) and CTGF (Figure 4) expression were corroborated by immunocytochemistry.
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Fibrous Cap Expression Phenotype
Only 25 genes were consistently differential in comparisons of fibrous cap pools (N=4), normal media (0.12% of genes examined), roughly half the frequency of differences observed for intima versus media, 11 were higher in the fibrous cap (Figure 2B; Table III, available online at http://atvb.ahajournals.org). The gene most highly expressed in cap SMCs versus media was connector enhancer of kinase suppressor of Ras 2 (CNKSR2) with a ratio of 6.9±2.0-fold. HLA-DRA, a known marker of immune activation in plaque SMCs and leukocytes, also marked cap from the media (5.4±1.1 SEM).24
Of the 14 genes more highly expressed in the media versus fibrous cap, the greatest ratio was noted for regulator of G protein signaling 5 (RGS5) at 5.9±1.3 SEM. We have previously shown this gene is highly expressed in normal aortic media versus vena cava at a ratio of 46.5±12.6 SEM and in arteries, but not veins throughout the vasculature.6,25 In contrast, RGS5 expression in the intima and the media were approximately equal.
When fibrous cap expression was contrasted with the nonatherosclerotic intima, 43 genes were consistently higher in the intima and 11 genes consistently higher in the cap (54 total, 0.26% of genes examined; Figure 2C and Table; Table IV, available online at http://atvb.ahajournals.org) including elevations in the intima of the artery marker RGS5. Other genes elevated in the intima included NMMHC-B, peptidyl amidating mono-oxygenase (PAM), S1–5 (S1–5/EFEMP1),6 and Veriscan and in fibrous cap the transcription factor Dp-1, pericentriolar material-1 (PCM1), and spleen tyrosine kinase (SYK). Intima expressed three markers higher relative to both media and fibrous cap: NMMHC-B, GTP-binding mitogen-induced T cell protein (GEM), and Down syndrome candidate region 1-like 1 (DSCR1L1). Cap was distinguished from both intima and media by downregulation of RGS5, alcohol dehydrogenase IB, and mitogen-activated protein kinase kinase kinase 10. In situ samples show loss of expression of RGS5 message (Figure 3) in the fibrous cap compared levels in to normal media and in intima, and immunohistochemistry shows the lack of versican expression in the fibrous cap compared with intima (Figure 5).
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Examination for Contamination of SMC Layers by RNA From Whole Plaques
For whole plaque comparisons, please see text in online supplement.
Sensitivity Analysis of Data
To ascertain whether the sizes of the identified data sets were specific to the proper placement of numerator and denominator tissues, we flipped one-half of each numerator/denominator pairings in each 2 tissue comparisons (except for whole plaque comparisons) and sorted the data as before for consistent genes (see Methods for details.). We previously performed this type of flipping or permutation analysis in our studies of PTFE graft neointima, producing random data sets of <5% of the size of the properly ordered data sets.7 There were no genes identified as differential between fibrous cap and intima; none between fibrous cap and media; and only 4 between intima and media when flipped. This shows that only the proper ordering of data into consistent numerator and denominator pairings produces the size data sets we are reporting in this study. This suggests that, because we would not obtain the data sets of consistent genes by chance from rearranged sets of array data, these gene sets are derived from actual biological differences rather than random noise derived from both system and biological variation.
Validation With Additional Intima and Media Samples
To test the predictive value of the 4 of 4 consistent data sets for defining the phenotypes of intima, cap, and media, we examined the expression of an additional pool of intima and cap samples. This approach is based on the leave-one-out strategy where a single additional sample is used to test the validity of the original analysis. Those genes consistent in 5 of 5 samples have a higher tissue predictive value versus the remainder of the set. In the comparison of a fifth pool of intima mRNA, intima-5, to caps, 32 of the 54 differentially expressed genes remained consistent, 23 intima and 9 cap markers. In a comparison of intima-5 versus media, 17 of the 48 differentially expressed genes remained consistent, 16 intima, including NMMHC-B, and 1 media marker, myosin-5C. In the comparison of cap-5 to intima, 41 of 54 markers remained consistent, 9 cap and 32 intima markers, including NMMHC-B and RGS5. In the comparison of cap-5 to media, 15 of 25 markers remained consistent, 9 cap and 6 media, including RGS5.
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Discussion |
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These array profiling data provide the first systematic analysis we are aware of for the phenotypic properties of either the fibrous cap or the nonatherosclerotic intima adjacent to the plaque. Intima is presumably the tissue within which plaques arise.26 The cap phenotype includes 11 genes that are consistently upregulated in the fibrous cap versus the intima and 43 genes that are consistently downregulated in the fibrous cap versus the intima. The expression ratios for these genes range from 12.8±2.3 SEM-fold to 1.8±0.5 SEM-fold. Perhaps most importantly, the expression profile of the fibrous cap is distinguishable from that of the underlying plaque and, more surprisingly, from the profile of the adjacent nonatherosclerotic intima. In addition, the absence of consistent matrix metalloproteinase (MMP) expression suggests that the cap itself is different from the shoulder region (which we removed from the cap edges) and perhaps more stable.27 Although we have verified RGS5, NMMHC-B, versican, and CTGF here by additional methods, lack of good probes and antibodies for ESTs has prevented extensive validation of these gene sets. However, we believe these EST data presented here will be valuable and interpretable to researchers in the future as these clones and clusters will become identified by bioinformatics advances as genes and their functions and biological importance become known as has been true for many of the newly annotated genes in the genome over the last few years. The reader will be able to link the accession numbers in our tables to genes with real functions and biological processes using a standard bioinformatics clustering search tool such as the National Center for Biotechnology Information’s Unigene genome clustering site (http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=9606). In addition, limited RNA availability and yield per sample has prevented the use of quantitative RT-PCR to measure fibrous cap mRNA levels. Future work with technologies for limited RNA samples will be required to fully validate expression in this and other array studies in disease processes when sample sizes are limiting.
It is intriguing that there were more consistent differences between the intima and the media than between the cap and the media, as the adjacent nonatherosclerotic intima appears much more media-like than does the cap. Both the intima and the media had more highly expressed gene markers versus the cap, and the intima had the highest number of differentially expressed genes versus the other two tissues, suggesting that the intima is the most expression-consistent tissue of these three layers. The cap had fewer markers versus both the media and the intima, suggesting either that this layer was more variable in expression than the intima and media, or the cap simply expresses fewer total genes, assuming the cap overexpressed gene set is the same consistent fraction of the total number of genes expressed as for intima and media. The media samples used here were younger than those of either the cap or the media. Although this age difference may possibly have affected the expression patterns observed, normal nonatherosclerotic media is the standard tissue for comparison to plaque lesions, and it is rare to collect uncomplicated normal media from aged patients for age-matched controls. It will likely be fruitful in the future to explore differences between both normal and pathological media and normal and injury-induced human intima. Another variable in this analysis is that all three media samples were from males, whereas the cap and intimal pools contained between 0% to 50% female samples. However, there was a pool of all male samples for intima, and there were both male and female whole plaque samples, eliminating this possible complicating factor for the profiles of these tissues, because differences had to be absolutely consistent in all pools. Samples were also pooled temporally without respect to clinical complications, such as diabetes, hyperlipidemia, hypertension, etc. Thus, the genes identified in this study are likely independent of such variables because of the random assignment of cases to pools.
Of most interest may be intimal genes that are downregulated in the fibrous cap as compared with the intima. Several are already of interest in vascular biology and disease, including: NMMHC-B, a marker for human intima28; versican, a proteoglycan implicated in accumulation of LDL in intima and plaque29; RGS5 (discussed below)6; PAM; and DSCR1L1. NMMHC-B is of particular interest because of its expression patterns in the plaque lesion and injured vessels28; it is upregulated specifically in the intima of both tissues versus the media and the plaque. PAM, which is expressed in the intima, is an enzyme that catalyzes the conversion of peptide hormones into active 
-amidated products. More than half of all peptide hormones require this modification before gaining bioactivity,30 including calcitonin, oxytocin, vasopressin, and PDGF.31 We previously found PAM to be a marker of artery over vein,6 suggesting it plays a role in normal arterial biology, presumably by amidating peptides to functional forms in the wall. DSCR1L1 is expressed higher in the intima than in either the media or the fibrous cap. This gene belonging to a 3-gene family of calcineurin phosphatase inhibitors that bind and inhibit the catalytic domain of calcineurin.32,33 Transgenic overexpression of calcineurin in mouse causes cardiac hypertrophy and death.34 Calcineurin activates NFAT by stimulating nuclear translocation and causes a complex set of downstream signaling events, such as GPCR and receptor tyrosine kinase signaling that function through calcium signals. Cyclosporine and FK-506 can inhibit calcineurin phosphatase activity, suggesting a role for other calcineurin inhibitors (eg, DSCR1L1) in tissue remodeling. DSCR1L1, therefore, may play a role in intimal pathophysiology by regulating calcineurin and NFAT signaling pathways. Interestingly, myosin 5C was upregulated in the media over the intima, suggesting possible differences in contractility between media and intima.
Genes higher in the cap over intima were Dp-1, ZNF24, SYK, and PCM1. PCM1 is highly expressed in G1 and S phases35 and often found fused with Janus-activated kinase (JAK) in hematologic malignancies.36 SYK is a nonreceptor protein tyrosine kinase crucial for low receptor IgG Fc and IgE receptor signaling and has been implicated as an inhibitor of breast cancer growth and metastasis.37 Our data also show a paucity of inflammatory genes in the intima and cap versus the media. We suspect this is a product of arrays measuring total expression from the adjacent nonatherosclerotic intima and that the whole fibrous caps versus looking at whole plaque using in situ or IHC, which would pick up small areas of inflammation and inflammatory gene expression in these two tissues.
Our data on whole plaque should be viewed with caution because of the complexity of plaque tissue. For example, some genes previously reported as characteristic of atherosclerotic plaque were inconsistent in this analysis. For example, CD68, a commonly used marker for the plaque macrophage,38 as well as osteopontin and eotaxin, genes known as plaque markers,39,40 were upregulated in some but not all plaques. These data may result from the fact that we were arraying whole plaque, and looking at overall levels of genes and macrophage RNA may be just a small overall percentage of the total sample. In situ and IHC studies can detect small areas of inflammation and are better methods for fine detail analysis compared with array analysis of whole samples; microscopic-aided laser dissection, however, could be used to isolate inflammation-rich plaque regions for array analysis in the future. This variability suggests that more detailed studies of plaque to plaque, or even intraplaque variation, will be useful. Recently, for example, Faber et al used subtraction suppression hybridization to identify two genes whose expression was unique to ruptured plaques.8 We would have discarded these genes as inconsistent in the present study, because they were not expressed universally in all plaques.
Finally, we are especially intrigued by the loss from the fibrous cap of RGS5, a highly consistent differential marker of arteries versus veins.6 RGS5 inactivates contractile and trophic GPCR signals by binding to G subunits and activating their intrinsic GTPase activity.41 Targeted overexpression of a related RGS family member in cardiomyocytes prevented remodeling of the heart—a feature that may be lost in the atrophic fibrous cap cells depicted in Figure 1.42,43 Recent studies have shown RGS5 may play an important role in vascular development and vessel formation.44,45 Many SMC vasoactive and hypertrophic receptors are GPCRs, including the angiotensin, endothelin, norepinephrine, serotonin, urotensin, and sphingosine receptors. Our data may open an obviously speculative hypothesis that pathologic properties of the fibrous cap may depend on loss of normal responses to agonist artery G protein–coupled reception.
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Acknowledgments |
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This work was supported by National Institutes of Health grants NIH PO1 HL03174, NIH 5R37 HL26405, NIH RO1 HL58083 (to L.D.A. and S.M.S.), and NIH 2RO1 HL57557–04 (to J.L. and R.L.G.). We thank Isa Werny, Colette Norby-Slycord, Jonathan McBride, Deanna Brown, and Melany Grogan for outstanding technical assistance and Sharon Lindsey for manuscript preparation.
Received March 3, 2005; accepted October 28, 2005.
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