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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:2023.)
© 2002 American Heart Association, Inc.

Atherosclerosis

Gene Expression Profiling of Apoptosis-Related Genes in Human Atherosclerosis

Upregulation of Death-Associated Protein Kinase

Wim Martinet; Dorien M. Schrijvers; Guido R.Y. De Meyer; Jeff Thielemans; Michiel W.M. Knaapen; Arnold G. Herman; Mark M. Kockx

From the Division of Pharmacology (W.M., D.M.S., G.R.Y.D.M., J.T., A.G.H., M.M.K.), University of Antwerp, Wilrijk and HistoGeneX (M.W.M.K.), Edegem, Belgium.

Correspondence to Mark M. Kockx, MD, PhD, Department of Pathology, A.Z. Middelheim, Lindendreef 1, B-2020 Antwerp, Belgium. E-mail mark.kockx{at}uia.ua.ac.be

Abstract

Objective— Apoptosis substantially affects the cellularity and integrity of atherosclerotic plaques. It remains, however, unclear which key regulatory genes are involved. In this study, cDNA expression arrays were used to analyze transcript levels of 205 apoptosis-related genes in human carotid endarterectomy specimens versus nonatherosclerotic mammary arteries.

Methods and Results— Seventeen genes with a 2- to 5-fold relative expression difference were identified. One of the most apparent changes in human plaques was the overexpression of death-associated protein (DAP) kinase (5-fold), a positive mediator of apoptotic cell death. Differential expression of DAP kinase mRNA in human plaques relative to mammary arteries was confirmed by quantitative reverse-transcription polymerase chain reaction. Western blotting and immunohistochemistry demonstrated enhanced levels of DAP kinase protein in the plaque with negligible expression in non-atherosclerotic vessels. DAP kinase was located predominantly in foam cells of smooth muscle cell (SMC) origin. Uptake of aggregated LDL by cultured aortic SMCs as well as exposure of SMCs to the short-chain acyl ceramide derivative N-hexanoyl-D-sphingosine (C₆-ceramide) upregulated DAP kinase both at the mRNA and protein level.

Conclusions— Our data demonstrate that cDNA array technology can identify novel genes that might participate in cell death pathways underlying atherogenesis.

Key Words: atherosclerosis • apoptosis • cDNA array • death-associated protein kinase • gene expression

Apoptosis has been identified as a prominent feature of advanced human atherosclerotic plaques.^1–3 The distribution of apoptosis within the plaque as determined by in situ end-labeling techniques (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation [ISNT]) is heterogeneous, being more frequent (1% to 2%) in regions with a high density of macrophages. Early lesions that mainly consist of smooth muscle cells (SMCs) present very little apoptosis (<0.1%), suggesting that apoptosis in atherosclerotic plaques is strongly related to macrophage infiltration. In recent years, several factors with proapoptotic activity have been identified in human atherosclerotic plaques. SMCs within human fatty streaks or advanced plaques show increased expression of the proapoptotic protein Bax.³ Furthermore, cytokines with proapoptotic potential, such as interleukin-1, tumor necrosis factor (TNF)-, and interferon (IFN)- are secreted by inflammatory cells and contribute to local cell death.¹ Together with macrophages and T lymphocytes, mast cells are present in the inflammatory infiltrate and initiate SMCs apoptosis by releasing chymase in the extracellular matrix.⁴ Upregulation of matrix metalloproteinases in human plaques may result in cell detachment from the matrix and apoptotic cell death (anoikis). Macrophages can also induce SMCs apoptosis via Fas/Fas-L interactions.⁵ Other substances that are known to induce apoptosis in the plaque include oxidized LDLs, oxysterols, and high levels of reactive oxygen species.^1,2

Despite much progress in our understanding of programmed cell death, the significance of apoptosis in atherosclerosis remains unclear.² Recently, it has been proposed that apoptotic cell death contributes to plaque instability, rupture, and thrombus formation.⁶ However, with the exception of lipid-lowering strategies,⁷ medical applications to modulate apoptosis in favor of plaque stability and/or regression have not been defined yet. Therefore, we need to better understand which key regulatory genes are involved in the cell death commitment pathways underlying atherogenesis. In this study, cDNA expression arrays were used to simultaneously quantify expression of 205 apoptosis-related genes. A strong upregulation of death-associated protein (DAP) kinase was found in advanced atherosclerotic plaques of carotid endarterectomy specimens as compared with nonatherosclerotic mammary arteries. Because DAP kinase is a positive mediator of apoptosis,^8–11 our results point to a novel factor stimulating cell death in human plaques.

Materials and Methods

Carotid Endarterectomy Specimens
Human carotid endarterectomy specimens (n=20) were obtained from patients (mean age=72±2 years, 81% men) with a carotid stenosis of >70%.³ One half of the specimens was fixed in 4% formalin within 2 minutes after surgical removal. The other half was frozen in liquid nitrogen to be used for RNA extraction and for Western blotting. For laser capture microdissection (LCM) experiments (see below), specimens were fixed in an alcohol-based fixative before paraffin embedding. Nonatherosclerotic mammary arteries (n=17) obtained during bypass surgery (mean age of patients=68±3 years, 57% men) were used as negative control samples and were manipulated similarly. The present study has been approved by the Review Board of the University of Antwerp.

Antibodies
The following mouse monoclonal antibodies were used: anti-DAP kinase (clone DAPK-55), anti-smooth muscle actin (clone 1A4), anti-ß-actin (clone AC-15), which were from Sigma; anti-CD68 (clone PG-M1) and anti-p53 (clone DO-7), which were from DAKO; and anti-Bax (clone 6A7) and anti-poly(ADP-ribose) polymerase-1 (clone C2 to 10), which were from PharMingen. Polyclonal rabbit anti-caspase-3 and anti-active caspase-3 antibody were obtained from Biosource and PharMingen, respectively. Polyclonal rabbit anti-TNF receptor 2 antibody was a gift from W.A. Buurman (University of Maastricht, The Netherlands). Goat anti-mouse and sheep anti-rabbit peroxidase–conjugated secondary antibodies were purchased from Jackson and DAKO, respectively.

cDNA Expression Arrays
Total RNA was isolated from carotid endarterectomy specimens (2 groups of 4 specimens, 75% men) and mammary arteries (1 group of 4 segments, 50% men) using the Trizol reagent (Invitrogen). RNA samples were further purified with the StrataPrep Total RNA Microprep Kit (Stratagene). Quality of RNA samples was verified by long-distance reverse transcription polymerase chain reaction (RT-PCR) using the SMART PCR cDNA synthesis kit and Advantage 2 PCR kit (both from ClonTech). Probe mixtures were synthesized by reverse transcribing 5 µg of total RNA using Superscript II reverse transcriptase (Life Technologies), cDNA synthesis primer mix (ClonTech), and [-³²P] dATP. Hybridization experiments were performed with the human apoptosis array (ClonTech). After extensive washes, the membranes were analyzed by PhosphorImaging (Molecular Dynamics). Data were analyzed using AtlasImage 2.0 software (ClonTech). Normalization of the signal intensity between two arrays was based on the overall value of all the genes on the arrays (global normalization). Weak signals were filtered out by applying a background-based signal threshold of 200%. To define differential gene induction, we used a 2-fold threshold value. Array reproducibility was determined using two independent assays.

Real-Time Quantitative RT-PCR
Relative abundance of mRNA species was assessed using the 5' fluorogenic nuclease assay (TaqMan). (For more information, please see online version of article, which can be accessed at http://atvb.ahajournals.org.)

Immunohistochemistry and DNA in Situ End Labeling
The immunohistochemical reactions were conducted by an indirect peroxidase antibody conjugate method.⁷ For the detection of oligonucleosomal DNA cleavage, a stringent TUNEL technique was used.¹²

Cell Culture
Human aortic SMCs (American Type Culture Collection) were grown in SMC basal medium (Clonetics) supplemented with human recombinant epidermal growth factor (0.5 ng/mL), insulin (5 µg/mL), human recombinant fibroblast growth factor (2 ng/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), gentamicin (50 µg/mL), amphotericin-B (50 ng/mL), and 5% fetal bovine serum. In some experiments, N-hexanoyl-D-sphingosine (C₆-ceramide, 30 µmol/L; Sigma), dissolved in dimethyl sulfoxide, was added to the culture medium. Cell death of C₆-ceramide–treated cells was detected by flow cytometry using an annexin V-PI kit (BD Biosciences). To induce foam cell formation, SMCs were incubated with aggregated LDL (agLDL, Sigma, 300 µg/mL) for 3 days in medium containing 10% lipoprotein-deficient serum (Sigma) and antibiotics (vide supra). Lipid peroxidation of LDL as determined by measuring thiobarbituric acid–reactive substances was low and approximated always the detection limit of the assay (1 µmol/L of malondialdehyde equivalents). AgLDL was made by vortexing the LDL solution for 4 minutes in Eppendorf tubes. To analyze lipid uptake, cells were fixed in formaldehyde 4% (10 minutes) and stained with oil red O.

Immunoblot Assays
Carotid endarterectomy specimens (n=5) and control samples (n=5) were analyzed by Western blotting as described.¹³

LCM
Sections (10 µm thick) of carotid endarterectomy specimens (n=3) and mammary arteries (n=3) were deparaffinized in toluol (2 x 3 minutes). Slides were washed with isopropylalcohol (1 minute), 70% ethanol (1 minute each), diethyl pyrocarbonate-treated water (1 minute), and rapidly stained with hematoxylin (15 seconds). Next, sections were washed with RNase-free water (1 minute), dehydrated with an ethanol gradient (70% ethanol for 1 minute, 90% ethanol for 1 minute, and 100% ethanol twice in 1-minute cycles), washed with xylene (5 minutes), and air dried (20 minutes). Single cells were microdissected by using the Pixcell II LCM system (Arcturus Engineering Inc.). For each specimen, approximately 2500 cells were isolated. Total RNA was prepared from microdissected cells by using the Absolutely RNA Nanoprep Kit (Stratagene).

Statistical Analysis
The 95% confidence interval for the relative expression of genes verified by real-time quantitative RT-PCR was calculated by the unpaired Student’s t test. To test whether the relative expression of DAP kinase and Bax in cultured SMCs was different from 1, the one-sample t test was used. A value of P<0.05 was considered significant.

Results

Comparative Gene Expression Profiling
Total RNA was isolated from pooled carotid endarterectomy specimens (2 groups of 4 specimens) and from segments of nonatherosclerotic mammary arteries (1 group of 4 segments). SMART cDNA synthesis and long-distance PCR followed by agarose gel electrophoresis revealed that the RNA samples had a normal size distribution of 0.5 to 6 kb. [-³²P]-Labeled cDNAs were hybridized to a human apoptosis array containing 200- to 500-bp DNA fragments, in duplicate, for 205 known apoptosis-related genes and 9 housekeeping genes. When using the same RNA preparation, this procedure resulted in a highly reproducible pattern of gene expression (<1% of genes with 2-fold differential gene expression). Moreover, comparison of both groups of endarterectomy specimens revealed similar gene expression profiles (<5% of genes with 2-fold differential gene expression). We observed meaningful hybridization signals (>2-fold above background threshold) for approximately 50% of the arrayed cDNAs (see online Figure I, which can be accessed at http://atvb.ahajournals.org). Housekeeping genes always showed strong hybridization signals whereas negative control spots never hybridized. Analysis of the hybridization patterns led to the identification of 17 genes that were differentially expressed between normal and diseased tissue (Table). Thirteen genes, including two housekeeping genes, with a moderate differential steady-state expression level of 2- to 4-fold, were identified in both groups of endarterectomy specimens as compared with the negative control group. Four candidates corresponding to DAP kinase, c-jun proto-oncogene, jun-B, and wee 1 Hu CDK tyrosine 15-kinase demonstrated a differential steady-state expression of approximately 5-fold. DAP kinase was upregulated in the endarterectomy specimens, whereas c-jun proto-oncogene, jun-B, and wee 1 Hu CDK tyrosine 15-kinase were downregulated. Furthermore, it is noteworthy that bcl-2–related protein A1 was uniquely expressed in carotid plaques. (For a complete list of genes with their respective expression data, please see the online supplemental data, which can be accessed at http://atvb.ahajournals.org.)

View this table: [in this window] [in a new window]   Table 1. Differentially Expressed Apoptosis-Related Genes in Atherosclerotic Plaques of Carotid Endarterectomy Specimens Versus Nonatherosclerotic Mammary Arteries

In an additional experiment, expression profiles of medial SMCs from carotid arteries and mammary arteries were compared to verify whether the latter could be used as a negative control for carotid plaques. Because normal media from carotid arteries could not be used, small margins of medial SMCs adjacent to carotid endarterectomy specimens were isolated by LCM. An equal number of SMCs from mammary arteries was obtained in the same way. cDNA amplification using SMART technology followed by array hybridization did not reveal significant expression differences of the genes listed in Table 1 and thus justified the interpretation of our array results.

Validation of Array Results by Real-Time Quantitative RT-PCR
Real-time quantitative RT-PCR confirmed upregulation of DAP kinase (14.9-fold, 95% confidence interval=1.7 to 127.1), caspase-1 (4.5-fold, 95% confidence interval=1.4 to 14.9), Bax (4.1-fold, 95% confidence interval=1.2 to 13.5), and PCNA (3.4-fold, 95% confidence interval=1.6 to 7.2) in carotid endarterectomy specimens (n=5) versus mammary arteries (n=5). However, RT-PCR did not show differential expression of TNF receptor 2 (0.8-fold, 95% confidence interval=0.2 to 2.6).

Overexpression of DAP Kinase in Human Atherosclerosis
Because we were mostly interested in proapoptotic genes that were upregulated in human plaques, we focused our further analysis on DAP kinase expression. In addition to DAP kinase mRNA, we observed a marked overexpression of DAP kinase in atherosclerotic plaques at the protein level. Western blots showed a protein band of the correct size (160 kDa) in crude extracts of carotid endarterectomy specimens, whereas in mammary arteries DAP kinase was hardly detectable (Figure 1). To further substantiate the DAP kinase protein expression data, immunohistochemistry was performed. Strong DAP kinase immunoreactivity was observed predominantly in foam cells of SMC origin in the fibrous cap of atherosclerotic plaques (Figure 2A and 2B). Immunoreactivity was found exclusively in the cytoplasm of the labeled cells. DAP kinase–positive SMCs were localized in regions of the plaque that contained lipid-laden SMCs with Bax immunoreactivity (Figure 2E). SMCs from the media or in nondiseased control vessels were negative for DAP kinase (Figure 2A and 2F). Macrophages in the plaque stained weakly or did not show immunoreactivity (Figure 2C). The majority of DAP kinase–positive cells (>95%) did not contain cleaved caspase-3 and were not labeled by the TUNEL technique (Figure 2D), indicating that they were not in the execution phase of apoptosis.

View larger version (13K): [in this window] [in a new window]   Figure 1. Western blot analysis of DAP kinase expression in crude cell lysates of nonatherosclerotic mammary arteries (n=5) and carotid endarterectomy specimens (n=5). ß-actin served as a loading control.

View larger version (139K): [in this window] [in a new window]   Figure 2. Immunohistochemical detection of DAP kinase in atherosclerotic plaques of human carotid endarterectomy specimens. A, Low-power photomicrograph of endarterectomy specimen showing DAP kinase expression in the plaque (Pl) but not in the media (M); B, double immunohistochemical staining for DAP kinase (brown) and -SMC actin (blue) in the plaque. Open arrowheads show that DAP kinase is expressed in lipid-laden SMCs; C, double immunohistochemical staining for DAP kinase (brown) and CD68 (blue) in the plaque. Only SMCs (CD68 negative) show DAP kinase expression (arrowheads); D, DAP kinase immunolabeling (blue) combined with TUNEL (brown) in the plaque. DAP kinase–positive cells were TUNEL negative; E, double immunohistochemical staining for DAP kinase (blue) and Bax (brown, arrows) in the plaque; F, double immunohistochemical staining for DAP kinase (brown) and -SMC actin (blue) in mammary arteries. SMCs are DAP kinase negative. Scale bar=100 µm (A) and 20 µm (B–F).

Overexpression of DAP Kinase in SMCs Treated With agLDL
To examine whether foam cell formation stimulated DAP kinase expression, aortic SMCs were cultured either in the presence or absence of agLDL (300 µg/mL). SMCs did not contain significant amounts of lipid, as shown by oil red O staining, but slowly transformed into foam cells when exposed to agLDL (see online Figure II, which can be accessed at http://atvb.ahajournals.org). After 3 days of treatment, most SMCs had accumulated large droplets of lipid in the cytoplasm. We then examined whether these lipid-laden SMCs overexpressed DAP kinase and/or other proapoptotic proteins. Western blots showed that SMCs did not express DAP kinase protein when cultured in the absence of agLDL. In contrast, strong upregulation of DAP kinase protein could be detected after 2 days of agLDL treatment (Figure 3A). DAP kinase expression was associated with enhanced levels of the tumor suppressor protein p53. We also observed a progressive increase in Bax and TNF receptor 2 expression during foam cell formation. Furthermore, it was demonstrated that agLDL could significantly induce (3-fold) both DAP kinase and Bax gene transcription (Figure 3B). Upregulation of DAP kinase and Bax in lipid-laden SMCs was not associated with cell death because cells did not contain cleaved caspase-3 (Figure 3A) or cleaved poly(ADP-ribose) polymerase-1. Moreover, cells remained adherent during foam cell formation and had a normal morphology.

View larger version (23K): [in this window] [in a new window]   Figure 3. Upregulation of DAP kinase in aortic SMCs after uptake of agLDL. A, Western blot analysis of SMCs before and after agLDL treatment. Initiation of apoptotic cell death was evaluated by cleavage of caspase-3. Caspase-3 was not cleaved after agLDL-treatment. As a positive control for apoptosis, SMCs were exposed to TNF- (30 ng/mL) and cycloheximide (CHX, 20 µg/mL) for 16 hours. B, Quantification of DAP kinase and Bax mRNA by real-time quantitative RT-PCR. *P<0.05, **P<0.01 vs untreated cells (one-sample t test).

Because agLDL in atherosclerotic lesions is enriched in ceramide¹⁴ and ceramide-induced apoptosis is mediated by DAP kinase,¹⁵ SMCs were also treated with C₆-ceramide. Both DAP kinase protein and mRNA increased significantly, reaching maximal levels after 6 hours of treatment (Figure 4). SMCs did not undergo apoptosis because they were annexin V and cleaved caspase-3 negative but showed a significant increase in propidium iodide staining after 24 hours of treatment (12.7±0.9% [treated] versus 2.9±0.1% [untreated]).

View larger version (20K): [in this window] [in a new window]   Figure 4. Upregulation of DAP kinase in aortic SMCs by C6-ceramide. Cells were exposed to 30 µmol/L C6-ceramide and harvested at the indicated time points. Levels of DAP kinase protein and mRNA were determined by Western blotting (A) and real-time quantitative RT-PCR (B), respectively. *P<0.05, ***P<0.001 vs untreated cells (one-sample t test).

Discussion

In this study, we applied cDNA expression arrays to analyze transcript levels of 205 genes that are associated with apoptotic cell death in human atherosclerotic plaques. We found 17 apoptosis-related genes, including 2 housekeeping genes, with a 2- to 5-fold difference in expression level between carotid endarterectomy specimens and nonatherosclerotic mammary arteries. Although gene expression profiling using cDNA array techniques seems to be an elegant way to identify novel genes or pathways that may contribute to features of atherosclerosis,¹⁶ some pitfalls and/or study limitations should be considered when using this type of technology for the identification of apoptosis-related genes in atherosclerosis. First, apoptosis is a major event in the pathophysiology of atherosclerosis in which many proteins are involved.^1–3 The small number of differentially expressed apoptosis-related genes, as well as the moderate differences in expression levels, indicate that most cell death proteins are regulated via posttranscriptional mechanisms, including protein-protein interactions, posttranslational modifications (eg, phosphorylation, proteolytic cleavage, glycosylation), and regulation of their subcellular localization, which cannot be detected with cDNA arrays. Additional proteomics-based studies could be performed to overcome this particular limitation of RNA analysis. Second, it is important to note that differences in gene expression patterns between normal and atherosclerotic vessels frequently result from the presence of different cell types. This may, for example, explain the differential expression levels of two housekeeping genes (HLAC and GAPDH) between both groups. Third, there is a potential risk of obtaining false-positive results if adequate control experiments, such as quantitative RT-PCR or Northern blots, are not implemented. However, cDNA arrays provide a powerful tool for systemic analysis of gene expression in atherosclerosis.^17,18

One of the most remarkable changes related to apoptosis in human atherosclerotic plaques was the overexpression of DAP kinase both at the mRNA and protein level. DAP kinase is a proapoptotic calmodulin-regulated serine/threonine kinase that carries interesting modules, such as a C-terminal death domain.⁸ The death-promoting effects of DAP kinase depend on its catalytic activity, its correct intracellular localization to actin microfilaments, and on the presence of the death domain.^9–11 According to recent evidence, DAP kinase mRNA increases before cell death, induced by IFN-⁹ or transient ischemia.¹⁹ In this study, we found that DAP kinase was overexpressed in lipid-laden SMCs of human plaques. Moreover, DAP kinase expression was stimulated in cultured aortic SMCs when cells were exposed to aggregated LDL or C₆-ceramide. Because the ceramide content of aggregated LDL in atherosclerotic lesions is 10- to 50-fold higher than that of nonaggregated plasma LDL,¹⁴ ceramide could be a potential trigger for DAP kinase expression both in vivo and in vitro. The difference between foam cells of macrophage or SMC origin in respect to DAP kinase expression is presently unclear. However, recent reports suggest that plaque macrophages develop several anti-apoptotic mechanisms, some of which are mediated by uptake of oxLDL or agLDL.²⁰ From this point of view, it is reasonable to assume that the absence of DAP kinase upregulation in macrophages from human plaques contributes to the long-term protection against cell death.

Considering the proapoptotic potential of DAP kinase in a wide array of apoptotic systems, including cell death triggered by IFN-, TNF-, Fas, and detachment of extracellular matrix,^8–10 which are common processes in atherosclerotic plaques,¹ there is a possibility that DAP kinase is involved in SMC death. Overexpression of DAP kinase in HeLA cells results in a significant reduction of viable clones, which favors this hypothesis.⁹ Interestingly, DAP kinase expression in atherosclerotic plaques is localized exclusively in the cytoplasm, which is a general prerequisite for DAP kinase–induced cell death.⁹ Our results demonstrate, however, that overexpression of DAP kinase in SMCs induced by uptake of agLDL or after exposure to C₆-ceramide did not initiate apoptosis and that the majority of DAP kinase immunoreactive cells in human plaques were TUNEL negative. Because DAP kinase is also involved in the formation of autophagic vesicles,²¹ overexpression of DAP kinase in human plaques may point to the induction of type II programmed cell death (autophagy), which is caspase-independent. Another possibility is that DAP kinase–overexpressing cells in atherosclerotic plaques are primed to die by apoptosis (type I programmed cell death) but that additional cell death stimuli are required to initiate the execution phase. Indeed, SMCs derived from atherosclerotic plaques show increased rates of apoptosis in culture compared with cells from normal vessels, even in the presence of serum survival factors, such as IGF-1 and PDGF.²² Although little is known about the mechanisms that regulate apoptosis in SMCs, recent studies have shown that the viability of SMCs depends on the balance between proapoptotic and antiapoptotic proteins. Plaque SMCs show, for example, increased expression of p53²³ and Bax³ but low levels of Bcl-2.²² Furthermore, the sensitivity of SMCs to apoptosis is mediated by differential expression of Fas-signaling molecules²⁴ and by the transcription factor nuclear factor-B that controls the expression of inhibitor of apoptosis protein-1.²⁵ Interestingly, our in vitro data demonstrate simultaneous upregulation of DAP kinase and Bax in lipid-laden SMCs. DAP kinase and Bax mRNA in cultured SMCs treated with agLDL were upregulated to the same extent (approximately 3-fold). In contrast, SMCs in human plaques showed a difference in DAP kinase gene expression, which was at least 3 times higher in comparison with Bax. This might indicate that additional factors in the vessel wall different from lipid uptake are responsible for DAP kinase gene induction. On the other hand, Levy-Strumpf and Kimchi²⁶ proposed that gene transcription may not be the major mode of DAP kinase regulation because DAP kinase is also regulated at the posttranslational level. Although the exact mechanism remains to be determined, it has been demonstrated that DAP kinase is able to regulate itself by autophosphorylation, whereas the intrinsic kinase activity is stimulated by Ca²⁺/calmodulin.⁹

The downstream effectors and the biochemical pathways involved in the proapoptotic actions of DAP kinase are not fully known. Recently, an interesting link between DAP kinase and p53-mediated apoptosis was established.²⁷ Because p53 expression is enhanced in agLDL-treated SMCs as well as in SMCs of human atherosclerotic plaques,²² upregulation of DAP kinase in atherosclerotic plaques may reflect one of the mechanisms underlying p53 activation. Interestingly, DAP kinase mRNA and protein levels are frequently lost in human cancer cell lines,²⁸ suggesting that DAP kinase controls tumor development. Because atherosclerotic plaques contain a large monoclonal population of SMCs,²⁹ they may be regarded as monoclonal benign neoplasms of the arterial wall. Cancer lesions do not arise from atherosclerotic plaques; therefore, it is plausible to assume that DAP kinase (or related proteins) in SMCs of the plaque plays an important role in cell growth control by augmenting the sensitivity of cells to cell death signals or by activating p53 as mentioned above.

In conclusion, our results demonstrate that cDNA array technology can be used to identify differentially expressed apoptosis-related genes in human atherosclerosis. We found overexpression of mRNA and protein for DAP kinase in human plaques. The present results support the need for further studies with DAP kinase knockout mice or studies with pharmacological inhibitors to clarify its contribution in atherogenesis and plaque destabilization.

Acknowledgments

This research was supported by the Fund for Scientific Research-Flanders (projects G.0080.98 and G.0180.01). M.M. Kockx held a fund for fundamental clinical research of the Fund for Scientific Research-Flanders. The authors are indebted to Martine De Bie for excellent technical assistance. Prof. Slegers (Cellular Biochemistry, University of Antwerp) is acknowledged for giving access to his phosphorimager.

Received July 9, 2002; accepted September 11, 2002.

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