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Gene Expression in Atherosclerotic Lesion of ApoE Deficient Mice

Molecular Medicine volume 7, pages 383–392 (2001)Cite this article

Abstract

Background

Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multi-factorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice.

Materials and Methods

ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters.

Results

The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice.

Conclusions

The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.

Introduction

Atherosclerosis is the major cause of mortality and invalidity in industrialized countries (1). The disease progresses over several decades and may remain silent until clinical manifestations occur, often with fatal outcome due to, for example, heart infarction or stroke. Although atherosclerosis is a multifactorial disease (1), it is highly correlated with high plasma cholesterol levels (2). Atherosclerosis has been described as an inflammatory disease due to the specific cellular and molecular responses at the site of lesions (2).

The inflammatory changes of the vessel wall recognized today are characterized by early expression of adhesion molecules on activated endothelium (3,4). Blood-derived mononuclear cells, mainly monocytes and T lymphocytes (T cells), extravasate through the endothelial layer (5,6). In the subendothelial space, monocytes differentiate into macrophages and start internalizing modified low-density lipoproteins (6) that have been captured by the extracellular matrix (7). Macrophages may present lipoprotein-derived antigens to T cells (8), that in turn secrete inflammatory cytokines such as interferon-γ (9). In advanced lesions, smooth muscle cells appear in the intima and form a fibrous cap over a necrotic core of the lesion (10).

Mouse models are increasingly used to explore the mechanisms in the pathogenesis of atherosclerosis (11). The ApoE-deficient (ApoE-/-) mouse has gained increasing interest as a suitable model of atherosclerosis and offers the unique possibility to evaluate the disease progress at different stages (12,13).

To increase the understanding of the pathogenesis of the disease, several candidate genes have been explored, although the methodological evaluation has previously been limited to a few genes. The recent development of gene array technology offers new possibilities to evaluate many genes at the same time. The evaluation of the gene expression patterns during the disease progress could lead to a better understanding of the key events in the pathogenesis of atherosclerosis (14).

In this study, we evaluated the gene expression in atherosclerotic ApoE-/- mice at 10 and 20 weeks on western diet applying a commercially available cDNA filter array.

Materials and Methods

Animals and Tissue Preparation

Female ApoE-/- mice (15) on the B6 background (strain C57BL/6H-ApoeTM1UNC129) were obtained from M&B Breeding and Research Centre (Bomholtgaard, Denmark) and normal C57BL/6 from Charles River Sverige AB (Uppsala, Sweden) at 6–8 weeks of ages. ApoE-/- mice were fed western diet containing 0.15% cholesterol for 10 or 20 weeks. Mice were sacrificed in groups of five mice by exsanguination under carbon dioxide anesthesia on consecutive days. After perfusion with ice-cold phosphate-buffered saline (PBS), the heart and the total aorta were dissected out and placed on ice-cold PBS. The samples were further rinsed mechanically under the dissection microscope before freezing. The aortas of five mice were pooled and stored at −808C until mRNA preparation. C57BL/6 mice fed normal chow were used as negative controls. Fifteen mice per group were compared.

Histologic Characterization of Atherosclerosis

The hearts were snap-frozen in n-heptane chilled with liquid nitrogen. Frozen cryostat sections were dried, fixed with 4% formaldehyde in PBS at room temperature for 10 min and rinsed in distilled H2O. After rinse in 60% isopropanol for 2 min, the samples were incubated in 0.67% Oil Red O for 15 min to visualize lipid deposits. Finally, all sections were counterstained with Harris’ hematoxylin.

mRNA Isolation, cDNA Synthesis, and Filter Hybridization

The frozen samples were homogenized in a dismembrator (B. Braun, Melsungen AG, Germany). Lysis buffer (Dynal, NY, USA) was added to the homogenate and mRNA isolated on oligo-dT-conjugated magnetic beads (Dynabeads, Dynal). The mRNA quantity was estimated using DNA Dip Stick (Invitrogen, Groningen, The Netherlands). The pooled mRNA from 3 3 5 mice (0.6 µg totally) from each group was precipitated with Na acetate. cDNA was labeled with [α-33P]dATP and hybridized to the mouse gene expression array (Clontech Laboratories Inc., Palo Alto, CA, USA) following the instructions of the manufacturer. The membranes for the two groups of each time were exposed on the same phosphor plate (Fuji BAS 2040, Fujifilm, Tokyo, Japan) for 4–14 hours and were quantified on a BAS 2500 Bio-Imaging Analyzer (Fujifilm).

Real-Time Polymerase Chain Reaction

Twenty nanograms of mRNA from each sample were reverse transcribed (RT) using superscript II according to the manufacturers manual (Gibco, Life technologies, Rockville, MD, USA). One and a half microliters of cDNA was amplified by real-time PCR with 1x TaqMan Buffer, 5mM MgCl2, 200µM of each dNTP, 200µM of each primer, 1.25pM of probe, 0.25U Amp-Erase Uracil N-Glycosylase, 1.25 U AmpliTaq Gold (PE Biosystems, Foster City, CA, USA). For the amplification of the iNOS gene (16), the primers iNOS-FW: 5-CAG CTG GGC TGT ACA AAC CTT-3 and iNOS-RV: 5-CAT TGG AAG TGA AGC GTT TCG-3 (GIBCO/BRL, Grand Island, NY, USA) and probe iNOS-TM: 5-CGG GCA GCC TGT GAG ACC TTT GA-3 (PE Biosystems) were used. For normalization of RNA loading between control samples were run using β-actin (16), the primers β-actin FW: 5-AGA GGG AAA TCG TGC GTG AC-3 and β-actin RW: 5-CAA TAG TGA TGA CCT GGC CGT-3 (GIBCO/BRL, Grand Island, NY, USA) and the probe β-actin TM: 5-CAC TGC CGC ATC CTC TTC CTC CC-3 (PE Biosystems) were used. Each sample was analyzed in duplicates (2 min at 508C, 10 min at 958C, 0.15 min at 958C, and 1 min at 608C) using ABI Prism 7700 Sequence Detector (PE Biosystems). The PCR amplification was correlated against a standard curve. The reactions were performed in MicroAmp Optical 96-Well Reaction Plates (PE Biosystems).

Data and Statistical Analysis

The image was imported in the Image Gauge Version 3.0 computer program (Fujifilm). The light intensity/cm2 was measured for one gene at a turn, applying exactly equal areas per gene for both groups at each time point. Background values for equal squares were taken at close locations on the filter and subtracted from the raw data. The gene expressions for each group at 10 and 20 weeks of treatment were subjected to regression analysis applying StatView 4.1 software.

Results

Experimental Set-Up

The experimental set-up was designed to analyze the differences between normal arterial walls and atherosclerotic lesions applying a commercially available mouse gene expression array (Clontech) that contained 588 genes. Atherosclerotic ApoE-/-mice were fed atherogenic western diet for 10 and 20 weeks and the gene expression was compared with age-matched C57BL/6 mice on normal chow. Because the lesion size of atherosclerotic plaques is very small in the mouse model, total aortas were dissected out, starting from the beginning of the aortic arch extending to and including the ileac bifurcation, for isolation of mRNA. The root of the aorta was frozen for immunohistologic characterization of the lesions. The extent of atherosclerosis has previously been found to correlate to the disease stages in the aorta (13). Figure 1 shows the extent of atherosclerosis in the root of the aorta in ApoE-/- mice after 10 and 20 weeks of western diet compared to C57BL/6 mice after 20 weeks.

Fig. 1
figure 1

The histology of the extent of atherosclerosis lesions is shown. The representative roots of the aorta for ApoE-/-mice at 10 and 20 weeks of treatment and a control C57BL/6 mouse at 20 weeks of treatment are depicted. The histologic sections were stained with Oil Red O.

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Array Analysis

The quality of the autoradiographic spots was evaluated and only spots with circle-round demarcation from the background were considered positive. Genes that did not meet this criterion in all four groups were considered as not expressed. Following the exclusion of nonexpressed or technically unreadable genes, 370 genes out of 588 genes remained for further analysis. To investigate whether the genes expressed in ApoE-/- mice correlated to the expression levels in C57BL/6 mice, intensities of the individual genes expressed in ApoE-/- mice were plotted in a regression analysis against the intensities of the individual genes expressed in C57BL/6 mice for 10 and 20 weeks of treatment (Fig. 2). At the first time point, 10 weeks, the overall gene expressions were highly correlated (R2 5.94) between the two groups at an interval of approximately three orders of magnitude and only a few genes visibly deviated from the main trend. At 20 weeks on diet, the correlation coefficient decreased (R2 5.902), as would be expected due to the progressive changes in atherosclerotic aortas of ApoE-/- mice, but still showed a high correlation. To normalize the individual genes, first the average gene expression intensities for each group were divided by the average of gene expression intensity of the C57BL/6 mice after 10 weeks of treatment. The expression intensities of the individual genes of the different groups were thereafter divided by the respective normalization factors.

Fig. 2
figure 2

Regression analysis of the genes expressed in ApoE-/- mice compared to C57BL/6 mice after 10 and 20 weeks of treatment. The scatter diagrams show the expressed genes of ApoE-/- mice versus C57BL/6 mice at 10 (left) and 20 weeks (right) of treatment and the regression line. The x- and y-axis show the signal intensity. The formula gives the value for the regression line.

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Gene Cluster Analysis

To analyze the gene expression over time, the ratios between ApoE-/- mice and C57BL/6 mice of the included genes at 10 and 20 weeks were calculated. To get a third point for the graphical visualization of the gene expression, the ratios between ApoE-/- and C57BL/6 mice for all genes were set to 1 (5 no difference) for a time point “0 weeks”; assuming that the gene expression of ApoE-/- mice and C57BL/6 mice would correlate even more strongly at the start of the study than after 10 weeks of treatment (Fig. 2), because the disease might not yet have accelerated. To visualize changes in gene expression over time, the web-based program GENECLUSTER (17) was applied; it organizes gene expression into patterns using self-organizing map (SOM) algorithms. A 4 3 2 SOM analysis resulted in eight clusters with different expression patterns (Fig. 3). As expected, a majority of genes showed only small changes in gene expression (Cluster 0, 1, and 2).

Fig. 3
figure 3

The gene expression profile during the development of atherosclerosis in ApoE-/- mice is shown in self-organizing maps (SOM). A 4 3 2 SOM summarizes the expression pattern of 370 genes using GENECLUSTER. Genes were submitted as ratios ApoE-/- versus C57BL/6 for 10 and 20 weeks and an imaginary ratio (5 1) as starting point for treatment (see results). The thick line indicates the mean of the ration of the genes in the cluster (n 5 number of genes per cluster) and the standard deviation. The thin lines indicate the minimum and maximum values.

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Genes summarized in Cluster 3 showed an increased expression in ApoE-/- mice at 10 weeks of diet whereas the gene ratios decreased to 1 or below at 20 weeks (Table 1). The genes from Clusters 4 and 6, showing a strong up-regulation at 20 weeks of diet in the ApoE-/- mice, are summarized in Table 2. Some genes were more than 10-fold up-regulated at that time point.

Table 1 Genes included in Cluster 3
Full size table
Table 2 Genes included in Clusters 4 and 6
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Genes from Clusters 5 and 7 are summarized in Table 3. These genes showed an increased expression in ApoE-/- mice at both time points, although not as strong at 20 weeks as genes from Clusters 4 and 6.

Table 3 Genes included in Cluster 5 and 7
Full size table

Quantitative RT-PCR

To validate the pattern and threshold expression levels of genes in the presented analysis we analyzed the expression of inducible nitric oxide sythetase (iNOS, Table 2), a gene with very low intensity in the array analysis, by quantitative real-time RT-PCR. Figure 4 shows RT-PCR values for iNOS. The gene expression of iNOS in the gene array showed a ratio of 0.68 and 2.4 in the ApoE-/- mice at 10 and 20 weeks, respectively. The quantitative RT-PCR showed a 14-fold and 63-fold increase in the ApoE mice at 10 and 20 weeks, respectively.

Fig. 4
figure 4

Quantitative RT-PCR of for iNOS, a gene with low expression values in the gene array. The evaluation of the expression levels for iNOS by quantitative RT-PCR is shown for all groups. The y-axis indicates the mRNA ratio of iNOS divided by the mRNA of β-actin. Mean 6 SEM of three pooled samples each containing five mice for each time point.

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Discussion

Our study included four groups of 15 animals that gave one analysis point. The high number of animals for each group should acceptably mirror the average gene expression for the different groups, although no statistical evaluation for each time point could be made. It was not feasible to undertake a study including replicates for each point due to the low yield of mRNA. However, regression analysis showed a very strong correlation between ApoE-/- and C57BL/6 gene expression at 10 weeks and a still good correlation for gene expression at 20 weeks. Reproducibility is likely. This suggestion is further supported by studies using commercial filter arrays from Clontech (18). Furthermore, the additional up-regulation of known atherosclerosis-related genes (see below) at 20 weeks compared to 10 weeks supports the reliability of the presented data. The genes that are only increased at 10 weeks of treatment must, however, be interpreted with caution.

To study the detection level of the array, we evaluated the expression of iNOS, a gene that previously has been associated with atherosclerosis (19) and showed low intensity levels in the array analysis. The array analysis and quantitative RT-PCR analysis showed an up-regulation of iNOS at 20 weeks on the diet. However, iNOS values at 10 weeks were lower for ApoE-/- than for C57BL/6 in the array analysis, but showed up-regulation in the quantitative RT-PCR. Thus, the gene array set-up used in this study was less sensitive for the analysis of iNOS expression than quantitative RT-PCR. This might be due to an underestimation of the ApoE-/- values for 10 weeks after normalization toward the average of the intensities of all included genes of the C57BL/6 group at 10 weeks of treatment.

Studies on gene expression of homogenous cell lines in vitro frequently define increases by a factor of 2 as positive up-regulation of gene expression. However, in our system, applying whole aortas to look at changes in gene expression of atherosclerotic tissue, we had to take into account that (i) the aorta of ApoE-/- mice included both diseased tissue as well as unaffected vessel wall, and (ii) that the diseased part in itself consisted of a heterogeneous mixture of different cell types. Thus, the changes we were looking for consisted only of a minute amount of total tissue or mRNA.

Atherosclerosis is a disease involving extravasation of blood-derived cells (5,6). Therefore, the changes of the atherosclerotic vessel wall in ApoE-/-mice should be reflected in the up-regulation of adhesion molecules as well as in novel signs of blood mononuclear cells when compared to nonatherosclerotic C57BL/6 mice. In agreement with this hypothesis, VCAM-1 (4) was up-regulated 3.83-fold in ApoE-/- mice at 10 weeks, and further increased to an 10.72-fold induction at 20 weeks compared to C57BL/6 mice. A similar pattern showed ICAM-1 (20) that increased from a 1.52-fold induction at 10 weeks to a 3.20-fold up-regulation at 20 weeks. A graphical illustration of the expression pattern of adhesion molecules present in Tables 1 through 3 is shown in Figure 5.

Fig. 5
figure 5

Expression pattern of adhesion molecules during the development of atherosclerosis in ApoE-/- mice are shown. The changes of expression for four adhesion molecules are depicted in the diagram. The y-axis indicates the ratios of gene expression between ApoE-/- mice and C57BL/6 control mice for 10 and 20 weeks of diet.

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Mac-1, a marker for macrophages, showed an expression level that increased from a 2.05-fold induction at 10 weeks, to an 8.8-fold induction at 20 weeks paralleling the increased influx of macrophages into the subendothelial space at sites of atherosclerosis. Additionally, CD14 and CD18, which are both expressed in monocytes/macrophages, increased from a 1.69- and 2.68-fold induction at 10 weeks to a 7.31- and 13.59-fold up-regulation at 20 weeks of treatment. Interestingly, the c-Fms proto-oncogene, encoding for the receptor of the macrophage colony-stimulating factor, which previously has been demonstrated to be up-regulated in human atherosclerotic lesions (21), showed a 1.15fold induction at 10 weeks that increased to 3.8-fold at 20 weeks. CD3, a marker for T cells, showed a 1.03-fold induction at 10 weeks and increased to a ratio of 2.00 at 20 weeks. Similarly, CD4 was up-regulated 1.1-fold (7.5 versus 7.0) at 10 weeks and 1.3-fold (8.6 versus 6.5) at 20 weeks (Cluster 2) in ApoE-/- mice compared to C57BL/6 mice. Additionally, CD40 that has evolved as an important signaling path in atherosclerosis (22), increased from a 1.14-fold induction to a 2.00-fold increase in ApoE-/-mice at 20 weeks of treatment.

Moreover, the increased expression of platelet-derived growth factor (1.19- and 1.60-fold induction at 10 and 20 weeks, respectively) might be associated with an increased proliferation and migration of smooth muscle cells from the media into the intima, a key feature of atherosclerosis (23,24). The expression of transcription factor egr-1 (Cluster 2) was increased at 20 weeks of treatment, although at a rather low level (a ratio of 0.99 and 1.3 at 10 and 20 weeks, respectively, when comparing the egr-1 expression in ApoE-/- and C57BL/6 mice). Egr-1 has recently been detected in human atherosclerotic lesions and confirmed in a related mouse model, the LDL receptor-deficient mouse (25). Phospholipase AII is expressed in our study in normal C57BL/6 mice and at increased levels in atherosclerotic ApoE-/-mice at 10 weeks (Table 1). These results are similar to the previous findings of the expression of phospholipase AII in normal and atherosclerotic human vascular tissue (26). Together, this indicates that ratios down to 1.13 might be interpreted as an up-regulation in our study.

In the present study, we compared ApoE-/- mice on a high cholesterol diet with C57BL/6 on normal diet to analyze the differences between atherosclerotic lesions and nonatherosclerotic vessels. We cannot exclude that some effects of gene expression may be due to compounds of the western diet (increased cholesterol levels), which are not due to development of atherosclerosis per se. However, the increases in expression of many genes associated with atherosclerosis over time imply that our experimental set-up truly mirrors atherosclerosis-related gene expression.

In addition to the established atherosclerosis-associated genes, several new candidate genes were found up-regulated in ApoE-/- mice. The macrophage chemoattractant protein-3 (MCP-3) (Table 3), a chemokine that attracts both macrophages and T cells, has previously been shown to be induced in vascular smooth muscle cells after cytokine stimulation (27). MCP-3 binds to the chemokine CC receptors-2 (CCR-2), similar to the related MCP-1, and CCR-3 (28). Interestingly, MCP-3 has additionally been suggested as an inhibitor of inflammation after cleavage by gelatinase 4 (29). Nerve growth factor (NGF) is up-regulated at 10 weeks and further increased at 20 weeks of treatment (Table 2). NGF is expressed in vascular smooth muscle in vascular remodeling after injury (30,31), but has not yet been evaluated in the context of atherosclerosis. Interestingly, extensive studies indicate that NGF might have an important role in inflammation (32–34). NGF and bFGF have been shown to increase mRNA levels of cathepsin-S, -B, and -L (35). These proteases were up-regulated during atherosclerosis in the present study and were confirmed by immuno-histochemistry in the lesions (Jormsjö S et al, manuscript submitted). CD41 T cells and mast cells might be a potential source for NGF in the atherosclerotic lesions (36,37). Hepatocyte growth factor (Table 2) has previously been shown to be present in rat and human vascular endothelial and smooth muscle cells (38). Hepatocyte growth factor has been shown to increase the expression of CD44 in endothelial cells and might thus contribute to adherence and extravasation of inflammatory cells (39,40). The overall expressions of both nerve growth factor and hepatocyte growth factor are low in C57BL/6, indicating that the up-regulation might be specific for the aortas of the ApoE-/- mice. Cellular retinoic acid binding protein-II (CRABP-II), a gene that is regulated by the retinoic acid receptor (RAR) signaling path (41), showed an increased expression and supports the previous finding of RARmediated signaling in human atherosclerosis (42). The low overall expression suggests a rather low retinoic acid mediated signaling in the control tissue (Table 3).

In conclusion, we studied gene expression during the development of atherosclerosis in the commonly used ApoE-/- mouse applying the commercially available Clontech mouse gene array. Having the drawbacks of our study design in mind, the gene expression in our study reflected features in the development of atherosclerosis that have been described earlier by others. Furthermore, the results indicate that gene expression is not a linear process with a maximal expression at advanced lesion stage. Rather, the pathogenesis should already to be evaluated in detail at early time points, to understand the development of atherosclerosis and to establish a preventive intervention. The present analysis is based on the evaluation of 377 genes and it will be necessary to interpret the present results in conjunction with future studies to evaluate the significance of the different findings.

References

  1. Braunwald E. (1997) Shattuck lecture-cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N. Engl. J. Med. 337: 1360–1369.

    Article  CAS  Google Scholar 

  2. Ross R. (1999) Atherosclerosis-an inflammatory disease. N. Engl. J. Med. 340: 115–126.

    Article  CAS  Google Scholar 

  3. Dong ZM, Chapman SM, Brown AA, Frenette PS, Hynes RO, Wagner DD. (1998) The combined role of P- and E-selectins in atherosclerosis. J. Clin. Invest. 102: 145–152.

    Article  CAS  Google Scholar 

  4. Cybulsky MI, Gimbrone MJ. (1991) Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251: 788–791.

    Article  CAS  Google Scholar 

  5. Hansson GK, Seifert PS, Olsson G, Bondjers G. (1991) Immunohistochemical detection of macrophages and T lymphocytes in atherosclerotic lesions of cholesterol-fed rabbits. Arterioscler. Thromb. 11: 745–750.

    Article  CAS  Google Scholar 

  6. Gerrity RG. (1981) The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am. J. Pathol. 103: 181–190.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Hurt E, Camejo G. (1987) Effect of arterial proteoglycans on the interaction of LDL with human monocyte-derived macrophages. Atherosclerosis 67: 115–126.

    Article  CAS  Google Scholar 

  8. Stemme S, Faber B, Holm J, Wiklund O, Witztum JL, Hansson GK. (1995) T lymphocytes from human atherosclerotic plaques recognize oxidized low density lipoprotein. Proc. Natl. Acad. Sci. U.S.A. 92: 3893–3897.

    Article  CAS  Google Scholar 

  9. Hansson GK, Holm J, Jonasson L. (1989) Detection of activated T lymphocytes in the human atherosclerotic plaque. Am. J. Pathol. 135: 169–175.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Newby AC, Zaltsman AB. (1999) Fibrous cap formation or destruction—the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation. Cardiovasc. Res. 41: 345–360.

    Article  CAS  Google Scholar 

  11. Lichtman AH, Cybulsky M, Luscinskas FW. (1996) Immunology of atherosclerosis: the promise of mouse models. Am. J. Pathol. 149: 351–357.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Breslow JL. (1996) Mouse models of atherosclerosis. Science 272: 685–688.

    Article  CAS  Google Scholar 

  13. Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. (1994) ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler. Thromb. 14: 133–140.

    Article  CAS  Google Scholar 

  14. Hiltunen MO, Niemi M, Yla-Herttuala S. (1999) Functional genomics and DNA array techniques in atherosclerosis research. Curr. Opin. Lipidol. 10: 515–519.

    Article  CAS  Google Scholar 

  15. Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. (1992) Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 89: 4471–4475.

    Article  CAS  Google Scholar 

  16. Overbergh L, Valckx D, Waer M, Mathieu C. (1999) Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11: 305–312.

    Article  CAS  Google Scholar 

  17. Tamayo P, Slonim D, Mesirov J, et al. (1999) Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation. Proc. Natl. Acad. Sci. U.S.A. 96: 2907–2912.

    Article  CAS  Google Scholar 

  18. Smid-Koopman E, Blok LJ, Chadha-Ajwani S, Helmerhorst TJ, Brinkmann AO, Huikeshoven FJ. (2000) Gene expression profiles of human endometrial cancer samples using a cDNA-expression array technique: assessment of an analysis method. Br. J. Cancer 83: 246–251.

    Article  CAS  Google Scholar 

  19. Detmers PA, Hernandez M, Mudgett J, et al. (2000) Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice. J. Immunol. 165: 3430–3435.

    Article  CAS  Google Scholar 

  20. Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R. (1998) Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler. Thromb. Vasc. Biol. 18: 842–851.

    Article  CAS  Google Scholar 

  21. Salomon RN, Underwood R, Doyle MV, Wang A, Libby P. (1992) Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma. Proc. Natl. Acad. Sci. U.S.A. 89: 2814–2818.

    Article  CAS  Google Scholar 

  22. Mach F, Schonbeck U, Libby P. (1998) CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 137(suppl):S89–S95.

    Article  CAS  Google Scholar 

  23. Wilcox JN, Smith KM, Williams LT, Schwartz SM, Gordon D. (1988) Platelet-derived growth factor mRNA detection in human atherosclerotic plaques by in situ hybridization. J. Clin. Invest. 82: 1134–1143.

    Article  CAS  Google Scholar 

  24. Murry CE, Bartosek T, Giachelli CM, Alpers CE, Schwartz SM. (1996) Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas. Analysis by competitive polymerase chain reaction. Circulation 93: 1095–1106.

    Article  CAS  Google Scholar 

  25. McCaffrey TA, Fu C, Du B, et al. (2000) High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. J. Clin. Invest. 105: 653–662.

    Article  CAS  Google Scholar 

  26. Hurt-Camejo E, Andersen S, Standal R, et al. (1997) Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries. Activity of the isolated enzyme on low-density lipoproteins. Arterioscler. Thromb. Vasc. Biol. 17: 300–309.

    Article  CAS  Google Scholar 

  27. Wang X, Li X, Yue TL, Ohlstein EH. (2000) Expression of monocyte chemotactic protein-3 mRNA in rat vascular smooth muscle cells and in carotid artery after balloon angioplasty. Biochim. Biophys. Acta 1500: 41–48.

    Article  CAS  Google Scholar 

  28. Kunkel SL. (1999) Through the looking glass: the diverse in vivo activities of chemokines [comment]. J. Clin. Invest. 104: 1333–1334.

    Article  CAS  Google Scholar 

  29. McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM. (2000) Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289: 1202–1206.

    Article  CAS  Google Scholar 

  30. Horiba M, Kadomatsu K, Nakamura E, et al. (2000) Neointima formation in a restenosis model is suppressed in midkine-deficient mice. J. Clin. Invest. 105: 489–495.

    Article  CAS  Google Scholar 

  31. Nemoto K, Fukamachi K, Nemoto F, et al. (1998) Gene expression of neurotrophins and their receptors in cultured rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 245: 284–288.

    Article  CAS  Google Scholar 

  32. Aloe L, Skaper SD, Leon A, Levi-Montalcini R. (1994) Nerve growth factor and autoimmune diseases. Autoimmunity 19: 141–150.

    Article  CAS  Google Scholar 

  33. Sanico AM, Stanisz AM, Gleeson TD, et al. (2000) Nerve growth factor expression and release in allergic inflammatory disease of the upper airways. Am. J. Respir. Crit. Care Med. 161: 1631–1635.

    Article  CAS  Google Scholar 

  34. Braun A, Appel E, Baruch R, et al. (1998) Role of nerve growth factor in a mouse model of allergic airway inflammation and asthma. Eur. J. Immunol. 28: 3240–3251.

    Article  CAS  Google Scholar 

  35. Liuzzo JP, Petanceska SS, Devi LA. (1999) Neurotrophic factors regulate cathepsin S in macrophages and microglia: a role in the degradation of myelin basic protein and amyloid beta peptide. Mol. Med. 5:334–343.

    Article  CAS  Google Scholar 

  36. Lambiase A, Bracci-Laudiero L, Bonini S, et al. (1997) Human CD41 T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors. J. Allergy Clin. Immunol. 100: 408–414.

    Article  CAS  Google Scholar 

  37. Leon A, Buriani A, Dal Toso R, et al. (1994) Mast cells synthesize, store, and release nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 91: 3739–3743.

    Article  CAS  Google Scholar 

  38. Nakamura Y, Morishita R, Higaki J, et al. (1995) Expression of local hepatocyte growth factor system in vascular tissues. Biochem. Biophys. Res. Commun. 215: 483–488.

    Article  CAS  Google Scholar 

  39. Hiscox S, Jiang WG. (1997) Regulation of endothelial CD44 expression and endothelium-tumour cell interactions by hepatocyte growth factor/scatter factor. Biochem. Biophys. Res. Commun. 233: 1–5.

    Article  CAS  Google Scholar 

  40. Adams DH, Harvath L, Bottaro DP, et al. (1994) Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells. Proc. Natl. Acad. Sci. U.S.A. 91: 7144–7148.

    Article  CAS  Google Scholar 

  41. Niforas P, Chu MD, Bird P. (1996) A retinoic acid/cAMP-responsive enhancer containing a cAMP responsive element is required for the activation of the mouse thrombomodulin-encoding gene in differentiating F9 cells. Gene 176: 139–147.

    Article  CAS  Google Scholar 

  42. Wuttge DM, Rommert A, Eriksson U, et al. Induction of CD36 by all-trans Retinoic Acid. Retinoic Acid Receptor Signaling in the Pathogenesis of Atherosclerosis. FASEB J 2001 March 20 [epub ahead of print]

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Acknowledgments

We are greatly in debt to Inger Bodin for technical assistance. This project was supported by grants from the Swedish Medical Research Council (12660), the Swedish Heart-Lung Foundation, the Torsten and Ragnar Söderberg Foundation, the Åke Wiberg Foundation, the Magnus Bergvall Foundation, the Foundation for Old Servants, and the Professor Nanna Svartz Foundation.

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Authors and Affiliations

  1. Cardiovascular Research Unit, Center for Molecular Medicine, Department of Medicine, L8:03, Karolinska Hospital, S-171 76, Stockholm, Sweden

    Dirk Marcus Wuttge, Allan Sirsjö & Sten Stemme

  2. Department of Medicine, Karolinska Hospital, King Gustaf V Research Institute, Stockholm, Sweden

    Per Eriksson

Authors
  1. Dirk Marcus Wuttge
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  2. Allan Sirsjö
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  3. Per Eriksson
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  4. Sten Stemme
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Correspondence to Sten Stemme.

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Wuttge, D.M., Sirsjö, A., Eriksson, P. et al. Gene Expression in Atherosclerotic Lesion of ApoE Deficient Mice. Mol Med 7, 383–392 (2001). https://doi.org/10.1007/BF03402184

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  • DOI: https://doi.org/10.1007/BF03402184

Keywords

Molecular Medicine

ISSN: 1528-3658