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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:2235.)
© 2003 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Gene Expression in Macrophage-Rich Inflammatory Cell Infiltrates in Human Atherosclerotic Lesions as Studied by Laser Microdissection and DNA Array

Overexpression of HMG-CoA Reductase, Colony Stimulating Factor Receptors, CD11A/CD18 Integrins, and Interleukin Receptors

Tiina T. Tuomisto; Anna Korkeela; Juha Rutanen; Helena Viita; Jan H. Bräsen; Mervi S. Riekkinen; Tuomas T. Rissanen; Kari Karkola; Zsolt Kiraly; Konrad Kölble; Seppo Ylä-Herttuala

From the A.I. Virtanen Institute (T.T.T., A.K., J.R., H.V., M.R., T.T.R., S.Y.-H.), Department of Medicine (S.Y.-H.), Gene Therapy Unit (S.Y.-H.), and Department of Pathology (K.K.), University of Kuopio, Kuopio, Finland; Franz Volhard Clinic, Charité, Humboldt University Hospital (J.H.B.), Berlin, Germany; Cardiovascular Surgery, University Hospital Eppendorf (Z.K.), Hamburg, Germany; Max-Delbrück-Centrum für Molekulare Medizin & Institut für Pathologie, Universitätsklinikum Charité (K.K.), Berlin, Germany.

Correspondence to Seppo Ylä-Herttuala, MD, PhD, FESC, Professor of Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala{at}uku.fi

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Inflammatory cells play an important role in atherogenesis. However, more information is needed about their gene expression profiles in human lesions.

Methods and Results— We used laser microdissection (LMD) to isolate macrophage-rich shoulder areas from human lesions. Gene expression profiles in isolated cells were analyzed by cDNA array and compared with expression patterns in normal intima and THP-1 macrophages. Upregulation of 72 genes was detected with LMD and included 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, interferon regulatory factor-5 (IRF-5), colony stimulating factor (CSF) receptors, CD11a/CD18 integrins, interleukin receptors, CD43, calmodulin, nitric oxide synthase (NOS), and extracellular superoxide dismutase (SOD). Several of these changes were also present in PMA-stimulated THP-1 macrophages in vitro. On the other hand, expression of several genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C-I and C-II, decreased.

Conclusions— Overexpression of HMG-CoA reductase in macrophage-rich lesion areas may explain some beneficial effects of statins, which can also modulate increased expression of CD11a/CD18 and CD43 found in microdissected cells. We also found increased expression of CSF receptors, IRF-5, and interleukin receptors, which could become useful therapeutic targets for the treatment of atherosclerotic diseases.

Key Words: atherosclerosis • genes • molecular biology • plaque • macrophages

	Introduction

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Macrophages are important for the development of atherosclerotic lesions. cDNA array analysis has become an important tool for studying gene expression patterns because it enables simultaneous analysis of hundreds of genes and can therefore be used to study molecular events during the disease process.¹ Gene expression patterns during foam cell formation have been studied in cell culture² and atherosclerotic events in animal models³ as well as in human samples.^4–6 However, cell culture and animal models may not fully reflect molecular events in human lesions. On the other hand, when analyzing entire atherosclerotic vessels, the heterogeneity of arterial tissue, which includes endothelia, smooth muscle cells (SMCs), macrophages, T cells, mast cells, and fibroblasts, makes gene expression patterns difficult to interpret. Recently, laser microdissection (LMD) techniques have enabled isolation of relatively pure cell populations from histological sections.⁷ Trogan et al⁸ used LMD to dissect foam cells from apoE-deficient mice to study macrophage-specific genes, but no studies of human lesions using LMD combined with DNA array have been reported.

See cover

We have used LMD to isolate macrophage-rich shoulder areas from human atherosclerotic lesions and compared their gene expression patterns to macroscopically normal diffuse intimal thickening (DIT) by a cDNA array. Expression of several genes, such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was increased in lesion macrophages. The results may help in the identification and validation of treatment targets for cardiovascular diseases.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue Samples
Fresh arterial samples (n=13) were obtained from organ donors (n=10) and fast autopsies (n=3) (Table 1). Some patients had received antihypertensive treatment, but none were on statin therapy. Macroscopically normal and atherosclerotic areas were dissected. Samples were divided into two parts: one part was immersion-fixed in 4% paraformaldehyde/15% sucrose (pH 7.4) and embedded in paraffin;⁹ the other part was frozen in liquid nitrogen and stored for RNA analysis or was frozen in isopenthane and embedded in OCT compound. According to histology, the samples were classified as normal (n=5) and atherosclerotic lesions (n=8).¹⁰ All studies were approved by the Ethics Committee of the Kuopio University Hospital.

View this table: [in this window] [in a new window]   TABLE 1. Tissue Samples Used for the Analyses

Histology and Laser Microdissection
Paraffin sections were used for immunostainings for macrophages (CD68), T cells (CD3), endothelia (CD31), and SMCs (HHF35; all monoclonal antibodies from Dako Co.).¹¹ Controls included incubations where primary antibodies were omitted and incubations with class- and species-matched irrelevant immunoglobulins.¹¹

For LMD, 7-µm-thick OCT sections were used. Sections were fixed in 70% ethanol and counterstained with hematoxylin/eosin and dehydrated in graded ethanol and xylene. Macrophage-rich shoulder areas, adjacent lesion cap areas containing no inflammatory cells, and areas of macroscopically normal DIT were dissected (Leica DM LMD system, Leica) from the same individuals.¹²

Cell Culture
Human monocytic THP-1 cells (ATCC TIB-202) were cultured in RPMI1640 medium according to ATCC instructions. Cells were stimulated with 0.1 µmol/L PMA (Sigma) to induce differentiation into macrophages and collected at indicated time points. THP-1 cells were also similarly activated with PMA and incubated with 100 µg/mL oxidized LDL (Ox-LDL) for 72 hours.¹³

RNA Isolation
Total RNA was isolated from LMD samples using the Strataprep total RNA microprep kit (Stratagene) and was extracted from cultured cells using Trizol reagent (Gibco BRL). RNA was pooled from two independent cell culture experiments at each time point.

DNA Array Analysis
RNA from LMD cells was quantified with the RiboGreen RNA quantitation kit (Molecular Probes). 100 ng RNA was used for amplification. Amplification was done with T7 polymerase using three cycles as described elsewhere.¹⁴ Probes were prepared from 2.0 µg of amplified RNA, random hexamers, and Cy3-dCTP or Cy5-dCTP. The probes were hybridized as described on the Wellcome Trust Sanger Institute web site (http://www.sanger.ac.uk) to microarrays (Sanger center Hver1.2.1) containing approximately 10000 clones from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium collection, representing approximately 6000 different genes. 50 µg of total RNA from cultured cells was labeled with Cy3-dCTP or Cy5-dCTP. All analyses were repeated three times, including the use of reversed fluorophores. Quantification of signals was performed with QuantArray software (GSI Lumonics), and the background was subtracted from the signal intensities. The linearity of the signal intensities was checked using GeneSpring software (Silicon Genetics) with M-A plot, where M represents the log ratio of the test and control samples and the A is the total log intensity of each spot. Because the data were linear (ie, blot displayed 45° rotation), normalization to the median of the signals was allowed, according to the protocol of the Finnish Center for Scientific Calculation.¹⁵ Signals were normalized using GeneSpring software by dividing each gene by the median of its measurements in all samples, and intensity ratios were calculated. Gene expression was considered significantly up- or downregulated when the intensity ratio test/control was 2.0 or 0.5, respectively. Statistical significance of the differences was calculated according to Claverie.¹⁶ To reach the 5% significance level ||Int_test-Int_control|| must be 2.8_control when analyzed in triplicates, where Int_test and Int_control are the averages of normalized signal intensities in test and control samples in repeated experiments and is the distribution of signal intensities in the control sample.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
DNAase treatment and cDNA synthesis were performed as described.¹⁷ Primers for HMG-CoA reductase were 5'-ACAGGATGCAGCACA GAATG-3' and 5'-CTTTGCATGCTCCTTGAACA-3'. The PCR reaction consisted of 25 cycles of +94°C for 1 minute, +60°C for 1 minute, and +72°C for 90 seconds. Similarly, RT-PCRs were performed for ß-actin (5'-CTCTTCCAGCCTTCCTTCCT-3' and 5-TCTGCTTGCTGATCCACATC-3'), interferon regulatory factor-5 (IRF-5) (5'-TCTTCTGCTTTGGGGAAGAA-3' and 5'-GATGGAG CTCCTTGAATTGC-3'), VEGF (5'-CCCACTGAGGAGTCCAAC AT-3' and 5'-GCGAGTCTGTGTTTTTGCAG-3'), nitric oxide synthase (NOS) (5'-GCTCTCCGTGTAGCCTGAAG-3' and 5'-ACCGCAAGAATGATGTCTCC-3') and extracellular superoxide dismutase (GC-SOD) (5'-GAGCTTCTCCTCTGCTCCAA-3' and 5'-GGGAAGATCGTCAGGTCAAA-3').

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of Genes Typical for Macrophages in LMD Lesion Cells
All arterial samples were immunocytochemically characterized for lesion type and the presence of SMCs, macrophages, endothelia, and T cells.¹¹ Typical examples of arterial samples are shown in Figure 1A through 1F. LMD was used to dissect macrophage-rich shoulder areas of the advanced lesions and macroscopically normal DIT from the same patients (Table 1). Examples of the LMD are shown in Figure 1G through 1J. The number of T cells in the dissected shoulder areas was less than 1 to 2%. SMCs represented <10% of the total cell population. The prevalence of foam cells was small (<5%) in the LMD shoulder fractions. LMD DIT samples were almost exclusively composed of SMCs with only sporadic macrophages. Gene expression patterns of the LMD samples were analyzed by cDNA array after the RNA was amplified. To validate the analysis we first looked at genes typical for macrophages. Array analysis detected upregulation of 33 genes that were previously connected to macrophages (Table 2). These genes included cathepsin G; several colony stimulating factor (CSF) and interleukin receptors; iNOS; a receptor for macrophage stimulating 1 receptor (c-met-related tyrosine kinase); RAB33A, which is a GTP binding protein restricted to monocytic lineage; a toll-like receptor that plays a role in interleukin signaling; integrins CD11a/CD18; and sialophorin (CD43). The expression of endothelial cell specific genes such as cadherin 5, endothelin 1, endoglin, and endothelial TEK tyrosine kinase, or T-cell specific genes, such as CD3, CD8, CD6, and CD27 antigens, were not significantly changed between the analyzed samples; the ratios varied from 0.78 to 1.2.

View larger version (59K): [in this window] [in a new window]   Figure 1. Representative examples of arterial samples. A, Macroscopically normal artery, immunostaining for SMCs (HHF35). B, Normal artery, immunostaining for macrophages (CD68), serial section. C, Advanced lesion, immunostaining for SMCs (HHF35). D, Advanced lesion, immunostainings for macrophages (CD68), serial section. E, Advanced lesion, immunostaining for T cells (CD3), serial section. F, Nonimmune control, serial section. G, HE-stained diffused intimal thickening (DIT) before laser microdissection (LMD). H, DIT after LMD. I-J, Inserts in C and D enlarged. I, HE-stained lesion before LMD. J, Lesion after LMD; macrophage-rich shoulder area was dissected. Arrow indicates internal elastic lamina. Bar 200 µm.

View this table: [in this window] [in a new window]   TABLE 2. Genes Typical for Macrophages Identified in LMD Analysis

Comparison of Gene Expression in LMD Lesion Cells and Cultured Macrophages
Table 3 presents 62 genes that had altered expression levels in LMD samples and were not previously connected to atherosclerosis as primary products of macrophages. Genes were classified into functional classes as described previously¹⁸ and compared with PMA-treated THP-1 macrophages. Table 3 lists genes where changes (either 2.0-fold or 0.5-fold change) in gene expression profiles were seen in both lesion LMD cells and in PMA-stimulated THP-1 macrophages. Among these were several genes that have a role in signal transduction, for example, serine/threonine protein kinase, calmodulin and adenylate cyclase 7, cytochromes, calponins, and extracellular SOD. Interestingly, HMG-CoA reductase, which is involved in cholesterol biosynthesis, was highly upregulated in lesion macrophage-rich areas. In addition, several KIAA proteins that had unknown functions were up- or downregulated. The expression of HMG-CoA reductase was confirmed by RT-PCR in LMD fractions and PMA-stimulated macrophages (Figure 2). However, HMG-CoA reductase was not induced in Ox-LDL-treated, lipid-loaded THP-1 cells. In addition, the induction of IRF-5, NOS, and extracellular SOD and the downregulation of VEGF were confirmed by RT-PCR (Figure 2).

View this table: [in this window] [in a new window]   TABLE 3. Gene Expression Changes in LMD Human Lesion Macrophage-Rich Areas vs Control Areas as Compared to Changes in PMA-Stimulated, THP-1 Derived Macrophages vs Unstimulated THP-1 Cells

View this table: [in this window] [in a new window]   TABLE 3. (Continued).

View larger version (48K): [in this window] [in a new window]   Figure 2. RT-PCR for (A) HMG-CoA reductase, (B) IRF-5, (C) VEGF, (D) NOS, and (E) extracellular SOD. 1, Control THP-1 cells; 2, PMA-stimulated THP-1 cells; 3, Ox-LDL (100 µg/mL; 72 hour)-stimulated THP-1 cells; 4, -RT control; 5, Blank; MW, molecular weight marker. The size of the PCR products are 191, 301, 298, 300, and 305 bp, respectively. ß-actin RT-PCR showed similar loading on each lane (data not shown). RT-PCR shows induction of HMG-CoA, IRF-5, NOS, and extracellular-SOD in PMA-stimulated THP-1 cells and downregulation of VEGF in PMA-stimulated cells as compared with unstimulated cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used LMD and gene expression profiling for the analysis of macrophage-rich areas in human atherosclerotic lesions. Previously, analysis of gene expression in tissue samples has been mostly based on in situ hybridization and immunocytochemistry¹⁹ because it has been difficult to isolate pure cell populations from histological sections. LMD enables the isolation of relatively pure cell populations from well characterized tissue samples.^7,12 It is important to note that these macrophage-rich areas also contained some T cells (<1% to 2%), and it is possible that some genes are derived from T cells. However, it can still be concluded that macrophage-rich areas were successfully isolated since (1) several genes typical for macrophages were identified, (2) T-cell content was low, (3) expression changes in endothelial cell and T-cell-specific genes were not significant, and (4) similar changes were found in vitro in PMA-stimulated THP-1 macrophages. When interpreting the results, it should be remembered that DNA arrays do not necessarily reflect regulation of transcription but tissue levels of mRNA (ie, transcriptosome or expression phenotype) at the time of tissue collection, whereas in cell cultures it is possible to analyze changes in expression profiles with time.

Upregulation of a total of 72 genes was identified; 33 genes were previously connected to atherosclerosis and 39 were not. Twenty-three genes were downregulated. LMD results are based on the amplification of a small amount of RNA from individual samples, followed by DNA array analysis and standardization of the gene expression ratios using the GeneSpring program. Every effort was made to avoid any systematic bias in the results. However, we cannot totally exclude the possibility of some bias or that some important genes were not picked up by the analysis.

Upregulation of several important genes was detected. One of these was HMG-CoA reductase, which is a key enzyme in cholesterol biosynthesis.²⁰ It was highly upregulated in LMD samples. The reason for the high expression level may be due to the proliferation and/or differentiation of macrophages because similar findings were obtained from PMA-stimulated THP-1 macrophages. Interestingly, it has been reported that HMG-CoA reductase in macrophages is not very sensitive to downregulation by cholesterol derived from LDL,²¹ which together with the low prevalence of lipid-loaded foam cells (<5%) in the analyzed LMD fractions may contribute to these findings. On the other hand, when THP-1 macrophages were incubated in the presence of Ox-LDL, downregulation of the HMG-CoA reductase was observed, which indicates that lipid-loaded THP-1 macrophages may not be an accurate model of macrophages present in shoulder areas. However, further studies are needed to clarify these issues.

It has been suggested that HMG-CoA reductase inhibitors, statins, have several beneficial effects that are not directly related to their cholesterol-lowering effects.²² Even though this hypothesis of the pleiotrophic effects of statins has not yet been fully tested, it is possible that macrophage functions in lesion shoulder areas may be attenuated by statins because of the high induction level of HMG-CoA reductase in these cells. HMG-CoA reductase also plays a role in the regulation of cell surface integrins CD11a and CD18, which bind to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. These integrins upregulated in LMD cells, and statins have been shown to downregulate CD11a and CD18 in cell culture.²³ In addition, HMG-CoA reductase also functions in cell movements mediated by CD43,²⁴ which was upregulated in LMD cells. Thus, DNA array analysis suggests that statin therapy may have favorable effects through these mechanisms, which should reduce monocyte-macrophage activity and trafficking in atherosclerotic lesions.

Other upregulated genes included CSF receptors, interleukin receptors, calmodulin, iNOS, extracellular SOD, and novel p53 target gene IRF-5. We have also previously demonstrated with DNA array analysis that some well known macrophage genes, such as apoE, TIMPs and VEGF receptor-2, were upregulated in lesion macrophages.⁶ Activation of these genes indicates that growth factor activities, inflammatory mediators, and oxidative stress play important roles in the microenvironment of lesion macrophages. On the other hand, expression of genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C1 and C2, were decreased, indicating less efficient vascular maintenance and increased susceptibility to thrombosis. It is important to note that genes associated with increased lipid uptake, such as scavenger receptors (1.7-fold induction), were not significantly upregulated in the LMD samples.

It is concluded that LMD can be used to dissect small cell populations from atherosclerotic lesions and that these cells can be used for DNA array analysis. Results showed that, among other genes, HMG-CoA reductase was highly upregulated in lesion macrophage-rich areas, which may explain some beneficial effects of statins not related to their lipid-lowering activity. Also, additional potential targets for therapy of cardiovascular diseases were identified.

	Acknowledgments

 
Acknowledgments

We thank Seija Sahrio, Anneli Miettinen, and Marja Poikolainen for technical assistance and the staff at the Kuopio University Hospital for the collection of human samples. This study was supported by grants from Finnish Academy, Sigrid Juselius Foundation, and Finnish Foundation for Cardiovascular Research.

Received August 4, 2003; accepted October 6, 2003.

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