Proteomic and Metabolomic Analyses of Atherosclerotic Vessels From Apolipoprotein E-Deficient Mice Reveal Alterations in Inflammation, Oxida
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动脉硬化血栓血管生物学 2005年第10期
From the Departments of Cardiac and Vascular Sciences (M.M., U.M., X.Y., L.L., S.F., Y.H., Q.X.) and Basic Medical Sciences (Y.-L.C., H.T., J.R.G.), St George’s, University of London, UK.
Correspondence to Dr Manuel Mayr, Department of Cardiac and Vascular Sciences, St George’s University of London, UK, Cranmer Terrace, London SW17 0RE, UK. E-mail m.mayr@sgul.ac.uk
Abstract
Objective— Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E–/– mice on normal chow diet.
Methods and Results— Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E–/– mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E–/– mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E–/– mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool.
Conclusion— Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.
Our study is a first attempt to show how changes in the proteome and the metabolome are reciprocally connected during atherogenesis and provides evidence that attenuated lesion formation in apolipoprotein E–/– mice is associated with reduced oxidative stress and successful recovery of the vascular energy pool.
Key Words: animal model ? apolipoprotein E ? atherosclerosis ? metabolomics ? oxidative stress ? proteomics
Introduction
The generation of apolipoprotein E-deficient (apolipoprotein E–/–) mice1,2 has been one of the most critical advancements in the elucidation of factors affecting atherogenesis. It is currently the most popular murine model in cardiovascular research and has revealed important insights into atherosclerosis. But despite a decade of research, there is still a need for sophisticated experimental techniques to obtain a more comprehensive understanding of the complex pathophysiology.3 Previous studies have revealed apolipoprotein E-related alterations in the transcriptome.4 However, simple deduction of protein expression from mRNA transcript analysis is insufficient5 and, importantly, provides no information on post-translational modifications, which are known to be instrumental in many human diseases.
We recently analyzed the proteomic profile of mouse arterial smooth muscle that was markedly influenced by mutational ablation of the protein kinase C delta gene.6 Our proteomic findings were translated into a functional context by combining proteomics with metabolomic techniques, under in vivo7,8 as well as in vitro conditions.6 This new research strategy allows us to decipher dynamic alterations of cellular proteins and metabolites revealing multiple facets of a single pathogenesis.9
In vascular research, proteomics and metabolomics are still in their infancies. Human umbilical cord endothelial cells and arterial and saphenous vein medial smooth muscle have been scantily characterized, but most attempts to apply proteomic techniques to human atheroma were jeopardized by the accumulation of serum proteins and the genetic heterogeneity of human samples, as summarized in a recent review article.9 Thus, we decided to use a mouse model, which offers the opportunity to analyze protein changes during various stages of atherogenesis under well-defined laboratory conditions and in animals with identical genetic background facilitating proteomic comparisons by limiting biological variation.
Materials and Methods
Mice
All procedures were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. Apolipoprotein E–/– mice on a C57BL/6 background were purchased from Jackson Laboratories (West Grove, Pa) and maintained in our laboratory. Mice were fed a normal chow diet containing 4.5% fat by weight (0.02% cholesterol) and kept on a light/dark (12/12-hour) cycle at 22°C, receiving food and water ad libitum.
Assessment of Lesion Formation
Aortas from 10-week-old, 12-month-old, and 18-month-old mice were dissected from the brachiocephalic trunk to the iliac bifurcation. Macroscopically, no lesions were observed on the aortic surface of 10-week-old apolipoprotein E–/– mice. Aortic lesions in 12-month-old mice were quantified by estimating the lesion-covered area on the aortic surface (as percent of surface area) and were classified as light (<10%), medium (10% to 30%), and severe (>30%). Disease severity was further verified by oil red O staining of the aortic root (measured as averaged lesion per cross-section of the aortic sinus). The frequency of light, moderate and severe lesions in 12-month-old apolipoprotein E–/– mice on normal chow diet was 40%, 50%, and 10%, respectively. At 18 months, most apolipoprotein E–/– mice had severe lesions in their arteries.
Proteomic and Metabolomic Analysis
For proteomic and metabolomic analyses, aortas were rinsed thoroughly with cold phosphate-buffered saline to remove any blood components and frozen immediately in liquid nitrogen. Aortas from both sexes were used in all experiments. A detailed methodology is available online (http://atvb.ahajournals.org). Protocols can be downloaded from our website (www.vascular-proteomics.com).
Standard Methods
Western blotting, immunohistochemistry, and enzymatic assays are available online at http://atvb.ahajournals.org.
Statistical Analysis
Statistical analysis was performed using the analysis of variance and Student t test. Pairwise comparisons between metabolites were performed using the Bonferroni/Dunn test. Results were given as means±SE. P<0.05 was considered significant.
Results
Proteomic Analysis
To analyze changes in the proteome, we created protein profiles of aortas by 2-dimensional (2D) gel electrophoresis. Aortas were derived from 10-week-old and 12-month-old apolipoprotein E+/+ and apolipoprotein E–/– mice. Average gels were obtained from at least 4 animals per group. A direct overlay is presented in Figure 1. Using a broad range pH gradient (pH 3 to 10 NL), 2-D gels comprised 1500 protein features. Differentially expressed spots are highlighted in color (orange and blue indicate an increase in aortas of apolipoprotein E+/+ and apolipoprotein E–/– mice, respectively). Numbered spots were excised and subject to in-gel tryptic digestion. Protein identifications as obtained by mass spectrometry are listed in Table I (available online at http://atvb.ahajournals.org). For spots marked with an asterisk (*), further proof of identification was obtained by tandem mass spectrometry (n=38; Table II, available online at http://atvb.ahajournals.org). Representative spectra are shown online.
Figure 1. 2D map of proteins expressed in aortas of 10-week-old apolipoprotein E+/+ and apolipoprotein E–/– mice. Protein extracts were separated on a pH 3 to 10NL IPG strip, followed by a 12% SDS polyacrylamide gel. Spots were detected by silver staining. The figure represents a direct overlay of average gels from apolipoprotein E+/+ and apolipoprotein E–/– vessels. Average gels were created from 4 single gels (total n=8). Differentially expressed spots are highlighted in color (orange and blue for apolipoprotein E+/+ and apolipoprotein E–/– vessels, respectively). Proteins identified by mass spectrometry are marked with numbers and listed in Table I.
To quantitatively monitor protein changes during atherogenesis, aortas of 12-month-old apolipoprotein E–/– mice were classified according to their atherosclerotic surface area in vessels with light (<10%), medium (10% to 30%), and severe atherosclerosis (>30%). Confirmation was provided by oil red O staining (Figure 2A). Cholesterol levels were significantly increased in all subgroups of chow-fed apolipoprotein E–/– mice compared with wild-type controls (P<0.001 ANOVA), but no correlation was observed between cholesterol levels and disease severity in 12-month-old apolipoprotein E–/– mice (449±49 and 421±127 mg/dL in animals with light and severe lesions, respectively), which is in line with previous reports.10 Quantitative data on protein changes during disease progression are summarized in Table III (available online at http://atvb.ahajournals.org).
Figure 2. Atherosclerotic lesions in apolipoprotein E–/– mice. A, Representative photographs of oil red O-stained sections from aortic roots, indicating lesions (red color) of 10-week-old apolipoprotein E+/+ and apolipoprotein E–/– mice and 12-month-old apolipoprotein E–/– mice with light, medium, and severe disease. Original magnification x100. B and C, Western blots probed with antibodies to albumin, immunoglobulins, and apolipoprotein A1. Note that albumin undergoes extensive fragmentation in advanced lesions (C) and that immunoglobulins were barely detectable in apolipoprotein E+/+ aortas, but abundant in apolipoprotein E–/– vessels (B).
Accumulation of Serum Components
As expected, macrophage markers (MAC2, CapG) increased, whereas SMC markers (SM22) decreased, and serum proteins accumulated with lesion progression, including fibrinogen, transferrin, and hemopexin. Interestingly, immunoglobulin deposits were barely detectable in apolipoprotein E+/+ mice, but abundant even in aortas of young apolipoprotein E–/– mice (Figure 2B), forming 2 charge trains of molecular masses 25 700 and 50 800 Da with isoelectric point values of 7.8 to 5.8 on 2D gels. Whereas further immunoglobulin deposition occurred during lesion progression, albumin was subject to extensive fragmentation within advanced atherosclerotic plaques (Figure 2C). Apolipoprotein A1 (apoA1), the major protein fraction of high-density lipoprotein, whose protective anti-oxidative role in the cardiovascular system is well-established, was significantly reduced in aortas of apolipoprotein E–/– mice (Figure 2B).
Increased Oxidative Stress
Besides revealing differences in protein expression, 2D gel electrophoresis has the potential to display differences in posttranslational modifications. Redox-active cysteines constitute the main antioxidative component of peroxiredoxins.11 This protein family represents a special type of peroxidase as the protein is the reducing substrate itself; on oxidative stress, the cysteine in the active site is either oxidized to cysteine sulfenic acid or overoxidized to cysteine sulfinic acid. Whereas the first modification is DTT-sensitive and therefore undetectable in 2-D gels, the latter modification is DTT-resistant and results in a charge shift toward a more acidic isoelectric point.12 Thus, peroxiredoxins are often encountered as doublet spots in 2-D and the ratio of oxidized to reduced protein is a reliable surrogate marker for oxidative stress.11,12
1-Cys peroxiredoxin (1-Cys prx), a novel antioxidant conferring protection against oxidative membrane damage,13 was almost exclusively present as a reduced (basic) isoform in apolipoprotein E+/+ aortas, whereas oxidation of 1-Cys prx was detectable in vessels of young apolipoprotein E–/– mice, resulting in decreased abundance of the reduced isoform (Figure 3A). Consequently, the ratio of oxidized to reduced 1-Cys prx was 15-times higher in young apolipoprotein E–/– mice compared with wild-type controls (0.58±0.18 versus 0.04±0.03; P<0.05). Surprisingly, it temporarily normalized in vessels harboring light lesions (0.10±0.06 versus 0.04±0.03, nonsignificant); however, overall, there appeared to be a linear relationship between the extent of oxidation of 1-Cys prx and the extent of lesion formation in aortas of 12-month-old apolipoprotein E–/– mice (Figure 3A).
Figure 3. Oxidative stress in apolipoprotein E–/– aortas. The spot pair corresponding to 1-Cys peroxiredoxin (1-Cys prx) is marked with an arrow (A). Numbers correspond to protein identities in Table I. Quantitative changes in expression of the oxidized and reduced form of 1-Cys prx during atherogenesis are shown below. Note that 1-Cys prx is predominantly present as reduced protein in apolipoprotein E+/+ vessels but is oxidized in apolipoprotein E–/– vessels. Expression pattern of malic enzyme supernatant (MOD-1) (B). *Significant difference from wild-type controls, P<0.05, ** P<0.01.
Antioxidant Defense
1-Cys prx is able to reduce peroxidized membrane phospholipids by using glutathione (GSH) as a reductant.13 Under oxidative stress, GSH is oxidized to GSSG and subsequently reduced by GSH reductase through the coupling reaction of NADPH to NADP. Strikingly, GSH reductase activity was found to be increased in aortas of young apolipoprotein E–/– mice (78.4±12.4 IU/L versus 37.9±1.4 IU/L; n=3; P<0.05) and the oxidation state of 1-Cys prx in 12-month-old apolipoprotein E–/– mice correlated to the expression pattern of the cytosolic isoform of malic enzyme (MOD-1), which generates cytosolic NADPH,14 providing reducing equivalents for lipid synthesis and GSH recycling (Figure 3B). Aortas harboring only light lesions demonstrated a prominent change in MOD-1 (Figure 3B) associated with decreased oxidation of 1-Cys prx (Figure 3A), lower levels of the oxidative stress-induced protein HO-1 (Figure 4A and 4B), and a trend to higher GSH concentrations compared with age-matched vessels with advanced disease (42±0.9 versus 31±0.8 nmol/g wet weight; n=3; P=0.10). In contrast, upregulation of antioxidant proteins was only detectable in advanced, but not early, stages of disease (Figure 4A). This is consistent with previously published mRNA data, reporting decreased antioxidant transcription in aortas of apolipoprotein E–/– mice at the onset of lesion formation.15
Figure 4. Antioxidants in apolipoprotein E–/– aortas. Western blot analysis to determine expression levels of antioxidant proteins in aortic tissues, including heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), catalase 1, and peroxiredoxin 1 (A). Note that HO-1 expression is higher in aortas with advanced atherosclerosis compared with age-matched vessels harboring only light lesions (B).
Enzymatic Changes
Among the differentially expressed proteins were several glycolytic enzymes, including triose phosphate isomerase, transketolase, glyceraldeyde-3-phosphate dehydrogenase, enolase, and phosphoglycerate mutase, as well as all 3 subunits of the pyruvate dehydrogenase complex, which accomplishes the irreversible step from glycolysis to the trichloroacetic acid (TCA) cycle by converting pyruvate to acetyl-coenzyme A (CoA). Changes of enzymes involved in glucose metabolism were accompanied by a reduction of cytoplasmic malate dehydrogenase, which is involved in the transfer of cytosolic NADH into mitochondria. Concomitantly, short chain-specific acyl-CoA dehydrogenases, responsible for the degradation of short chain fatty acids to acetyl-CoA, were differentially expressed in aortas of young apolipoprotein E–/– mice and medium chain-specific acyl-CoA dehydrogenases became upregulated in vessels of old apolipoprotein E–/–mice (Table III).
Metabolite Changes
To clarify the metabolic net effect of these enzymatic changes, we measured vascular metabolites by high-resolution nuclear magnetic resonance spectroscopy. A representative proton magnetic resonance spectrum of an aortic extract is shown in Figure 5. Quantitative data are included as Table IV (available online at http://atvb.ahajournals.org), whereas Figure 6 shows a histogram displaying the relative metabolite ratios for apolipoprotein E–/– aortas derived from 10-week-old and 18-month-old apolipoprotein E–/– mice compared with wild-type controls. Decreased concentrations of alanine, a transamination product of pyruvate, were associated with a reduction of the adenosine nucleotide pool in aortas of young apolipoprotein E–/– mice and a coordinated but nonsignificant decline of other energy metabolites, such as total creatine and succinate, the oxidation of which is directly associated with respiratory chain reactions. The ratio of alanine to pyruvate was significantly decreased in young apolipoprotein E–/– mice compared with wild-type controls (1.7±0.8 versus 7.5±1.6; P=0.002) and remained reduced in old apolipoprotein E–/– mice (4.1±1.7 versus 7.5±1.6, P=0.019, respectively), but the adenosine nucleotide and creatine pool normalized. The metabolic profiles also revealed a significant increase in choline in aortas of old apolipoprotein E–/– mice. Interestingly, concentrations of trimethylamine oxide, a breakdown product of choline, were significantly higher in male than female aortas, suggesting a gender-specific difference in choline metabolism, which was independent of the apolipoprotein E genotype (inset in Figure 6). An additional comparison of metabolic profiles obtained from sex-matched aortas of 12-month-old apolipoprotein E–/– mice (n=3 in each group, 2 males, 1 female) revealed that aortas harboring only light lesions had a 1.7- and 1.9-fold increase in adenosine nucleotides (P=0.028) and succinate (P=0.060), respectively, but only half the glucose concentration (P=0.109) compared with aortas with severe disease.
Figure 5. Nuclear magnetic resonance spectra of a murine aorta derived from 18-month-old apolipoprotein E–/– mice. Within the aliphatic region (–0.05 to 4.2 parts per million) of the nuclear magnetic resonance spectra, resonances have been assigned to lactate (Lac), alanine (Ala), pyruvate (Pyr), acetate (Acet), succinate (Succ), carnitine (Car), choline (Cho), phosphocholine (PC), taurine (Tau), scyllo-inositol (Scy-ino), glycolic acid (Glyco), trimethylamine oxide (TMAO), glutamate (Glu), creatine (Cr), phosphocreatine (PCr). ADP+ATP and formate are showing in the aromatic region of the spectra (6.0 to 9.0 parts per million, see inset).
Figure 6. Comparison of metabolites in apolipoprotein E+/+ and apolipoprotein E–/– aortas. Relative changes of metabolites in aortas derived from 10-week-old (gray bars) and 18-month-old (black bars) apolipoprotein E–/– aortas compared with apolipoprotein E+/+ aortas (reference line). Abbreviations for metabolites are explained in the legend to Figure 5. Data are provided in Table IV. The inset highlights a gender difference for TMAO concentrations in murine aortas. *Significant difference with Bonferroni/Dunn, P<0.017.
Discussion
Our study provides evidence that immune activation, oxidative stress, and energetic impairment are among the earliest alterations in hyperlipidemic animals.
Inflammation
Immunoglobulin deposition within the vessel wall of apolipoprotein E–/– mice is close to peak levels even before lesion formation initiates and cannot be accounted for by impaired endothelial barrier function, because other serum components, such as albumin and fibrinogen, did not accumulate in vessels without overt atherosclerosis. In murine models, antibodies recognizing oxidized phospholipids correlate closely with lesion progression and regression16–18 and a class shift from IgG2a to IgG1, indicative for a switch of the T-cell response from Th1 to Th2, has been observed for circulating oxidized low-density lipoprotein antibodies in apolipoprotein E–/– mice.19 Similarly, we observed a preponderance of IgG1 within atherosclerotic lesions and mass spectrometry data obtained from the variable region of accumulated immunoglobulins suggest that at least some are directed against phosphocholine (gi30720232, anti-phosphocholine immunoglobulin heavy chain variable region [mus musculus], sequence coverage 33%). However, further studies will be required to allow for a more detailed characterization.
Oxidative Stress
Oxidative stress, the local imbalance between the ubiquitous formation of reactive oxygen species (ROS) and the equally ubiquitous antioxidant defenses, is thought to play an important role in vascular injury and atherogenesis.20–22 The complexity of the antioxidant defense have made it difficult to assess their impact on atherosclerosis as it is likely that knockout of individual ROS-generating or ROS-scavenging enzymes are compensated for by synergistic ones.23 Because the pathogenetic outcome is determined by the balance between pro-oxidants and antioxidants, measurements of individual enzymes at a single time are unlikely to shed much light and a more comprehensive approach is needed.23
Our proteomic data support the role of oxidative stress in atherogenesis: First, oxidation of 1-Cys prx, a reliable in vivo marker of oxidative stress,11,12 was significantly elevated in young apolipoprotein E–/– mice compared with wild-type controls. Second, the oxidation state of 1-Cys prx correlated to lesion size in aortas of 12-month-old mice indicating that reduced oxidative stress might attenuate lesion progression in apolipoprotein E–/– mice. Third, the observed reduction of oxidative stress in vessels with light lesions was not a result of increased expression of antioxidants, because protein levels of catalase 1, SOD-1, and peroxiredoxin 1 were similar to those in young apolipoprotein E–/– mice. The oxidation status of 1-Cys prx, however, showed a striking correlation to proteomic changes of malic enzyme supernatant (MOD-1). As demonstrated previously, such changes in the protein pattern are likely to reflect alterations in enzymatic activity.6–9 The soluble form of malic enzyme is 1 of 3 enzymes, apart from glucose 6-phosphate dehydrogenase and cytoplasmic isocitrate dehydrogenase, that can generate cytosolic NADPH,14 providing reducing equivalents for lipid synthesis, as well as for glutathione and thioredoxin reductase, which are of paramount importance in maintaining the reducing intracellular environment.24,25 The importance of thiol-based defense mechanisms in hyperlipidemia is supported by the increase in glutathione reductase activity in vessels of young apolipoprotein E–/– mice. This initial glutathione defense appears to be overwhelmed in advanced stages of disease as indicated by a rebound in the oxidation of 1-Cys prx, increased expression of HO-1, and lower glutathione levels compared with aortas harboring light lesions. Thus, upregulation of antioxidant proteins appears to be the last resort, once other counter-regulatory mechanisms cannot provide sufficient reducing equivalents to antagonize ROS, rather than the first attempt to confine oxidative stress. These findings would be consistent with previous studies reporting a weak glutathione-related enzymatic antioxidant shield in human atheroma.26
Despite a conjunct upregulation of antioxidant proteins in advanced stages of disease, deleterious consequences of reactive oxygen species became apparent, such as proteolysis of oxidatively damaged proteins.27 Albumin is degraded 50-times faster on oxidation,27,28 providing a possible explanation for its extensive fragmentation. Similarly, enzymes known to be susceptible to free radical-mediated inactivation such as aconitase and the Rieske protein of ubiquinol cytochrome C reductase, which contain iron-sulfur centers, a coordination complex with cysteine sulfurs of proteins, were altered in advanced stages of atherosclerosis.29,30 Such interactions are of potential importance, as damage to such complexes results in release of free iron and subsequent formation of hydroxyl radical, a highly reactive oxygen species, which perpetuates the vicious cycle of oxidative stress.31
Energy Metabolism
Metabolic disturbances are likely to be a key factor in both the initiation and progression of atherosclerosis. The upregulation of acyl-CoA dehydrogenases, the decrease in alanine, a transamination product of pyruvate, and the downregulation of cytoplasmic malate dehydrogenase, responsible for transferring cytosolic NADH produced during glycolysis into mitochondria,32 suggest that vascular cells might respond to hyperlipidemia by metabolizing lipids instead of glucose. Increased fatty acid oxidation would exert a negative feedback on the activity of the pyruvate dehydrogenase complex32 slowing down glucose metabolism, the main source of energy for the vasculature.33 Moreover, when excess fatty acids reach mitochondria, there is even a risk of uncoupling oxidation from phosphorylation with oxygen wastage.32 Insufficient phosphorylation of energy metabolites will cause their degradation and tissue depletion, providing a possible explanation for the observed reduction of the adenosine nucleotide pool in young apolipoprotein E–/– mice. Notably, breakdown products of adenosine are xanthine and hypoxanthine, both substrates for the xanthine oxidase, which is a powerful enzyme in the generation of ROS.34 It is noteworthy that the depletion of vascular energy metabolites coincided with increased oxidation of 1-Cys prx in young apolipoprotein E–/– mice, whereas attenuated lesion formation in 12-month-old apolipoprotein E –/– mice was associated with reduced oxidative stress and successful recovery of the adenosine nucleotide pool possibly serviced by increased glucose use. Supporting our findings are previous observations that insulin supplementation reduces lesion formation and oxidative stress in apolipoprotein E–/– mice35. In contrast, overexpression of the uncoupling protein 1 results in mitochondrial dysfunction and promotes atherosclerosis by depleting energy stores and increasing superoxide production.36 Thus, there is evidence that inefficient glucose and energy metabolism may contribute to oxidative stress and vascular disease in hyperlipidemic mice.
Study Limitation
A main obstacle for applying proteomic analysis to vascular pathology is the heterogeneous cellular composition of atherosclerotic plaques. Whereas smooth muscle cells dominate proteomic profiles of normal vessels, advanced lesions contain large numbers of monocyte-derived macrophages. Overall, the proteomic profiles were remarkably consistent in young and old apolipoprotein E–/– mice: only 2 macrophage proteins, namely CapG, which accounts for 0.6% of total macrophage proteins, and MAC-2, which is also abundant in activated macrophages, showed a significant increase in advanced stages of disease, indicating that the concentration of other macrophage proteins in aortic extracts was not high enough to allow detection on 2D gels. Thus, the proteomic and metabolic profiles remain dominated by vascular smooth muscle cells, facilitating data interpretation.
Finally, we should point out that our proteomic analysis revealed differential expression of several signaling proteins, but the vascular function of some proteins, eg, dihydropyrimidinase-like proteins 2 and 3, which are regulators of neuronal development and axonal outgrowth,37 is currently unknown. For others, there is evidence for their involvement in atherosclerosis: downregulation of Ras suppressor protein 1, an endogenous inhibitor of the Ras signaling pathway, during lesion progression, is consistent with a study reporting attenuation of lesion formation in apolipoprotein E–/– by inhibiting the Ras signaling pathway;38 the functional relevance of 14-3-3 gamma, an inhibitor of the protein kinase C signaling pathway, is supported by our findings that deficiency for protein kinase C delta accelerates neointima formation in a mouse model of vein graft arteriosclerosis.39
Summary
Our study is a first attempt to show how changes in the proteome and the metabolome are reciprocally connected during atherogenesis and provides evidence that attenuated lesion formation in apolipoprotein–/– mice is associated with reduced oxidative stress and successful recovery of the vascular energy pool.
Acknowledgments
The use of the facilities of the Medical Biomics Centre at St. George’s, University of London, is gratefully acknowledged.
This work was supported by grants from the British Heart Foundation and the Oak Foundation.
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Leitges M, Mayr M, Braun U, Mayr U, Li C, Pfister G, Ghaffari-Tabrizi N, Baier G, Hu Y, Xu Q. Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice. J Clin Invest. 2001; 108: 1505–1512.(Manuel Mayr; Yuen-Li Chun)
Correspondence to Dr Manuel Mayr, Department of Cardiac and Vascular Sciences, St George’s University of London, UK, Cranmer Terrace, London SW17 0RE, UK. E-mail m.mayr@sgul.ac.uk
Abstract
Objective— Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E–/– mice on normal chow diet.
Methods and Results— Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E–/– mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E–/– mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E–/– mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool.
Conclusion— Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.
Our study is a first attempt to show how changes in the proteome and the metabolome are reciprocally connected during atherogenesis and provides evidence that attenuated lesion formation in apolipoprotein E–/– mice is associated with reduced oxidative stress and successful recovery of the vascular energy pool.
Key Words: animal model ? apolipoprotein E ? atherosclerosis ? metabolomics ? oxidative stress ? proteomics
Introduction
The generation of apolipoprotein E-deficient (apolipoprotein E–/–) mice1,2 has been one of the most critical advancements in the elucidation of factors affecting atherogenesis. It is currently the most popular murine model in cardiovascular research and has revealed important insights into atherosclerosis. But despite a decade of research, there is still a need for sophisticated experimental techniques to obtain a more comprehensive understanding of the complex pathophysiology.3 Previous studies have revealed apolipoprotein E-related alterations in the transcriptome.4 However, simple deduction of protein expression from mRNA transcript analysis is insufficient5 and, importantly, provides no information on post-translational modifications, which are known to be instrumental in many human diseases.
We recently analyzed the proteomic profile of mouse arterial smooth muscle that was markedly influenced by mutational ablation of the protein kinase C delta gene.6 Our proteomic findings were translated into a functional context by combining proteomics with metabolomic techniques, under in vivo7,8 as well as in vitro conditions.6 This new research strategy allows us to decipher dynamic alterations of cellular proteins and metabolites revealing multiple facets of a single pathogenesis.9
In vascular research, proteomics and metabolomics are still in their infancies. Human umbilical cord endothelial cells and arterial and saphenous vein medial smooth muscle have been scantily characterized, but most attempts to apply proteomic techniques to human atheroma were jeopardized by the accumulation of serum proteins and the genetic heterogeneity of human samples, as summarized in a recent review article.9 Thus, we decided to use a mouse model, which offers the opportunity to analyze protein changes during various stages of atherogenesis under well-defined laboratory conditions and in animals with identical genetic background facilitating proteomic comparisons by limiting biological variation.
Materials and Methods
Mice
All procedures were performed according to protocols approved by the Institutional Committee for Use and Care of Laboratory Animals. Apolipoprotein E–/– mice on a C57BL/6 background were purchased from Jackson Laboratories (West Grove, Pa) and maintained in our laboratory. Mice were fed a normal chow diet containing 4.5% fat by weight (0.02% cholesterol) and kept on a light/dark (12/12-hour) cycle at 22°C, receiving food and water ad libitum.
Assessment of Lesion Formation
Aortas from 10-week-old, 12-month-old, and 18-month-old mice were dissected from the brachiocephalic trunk to the iliac bifurcation. Macroscopically, no lesions were observed on the aortic surface of 10-week-old apolipoprotein E–/– mice. Aortic lesions in 12-month-old mice were quantified by estimating the lesion-covered area on the aortic surface (as percent of surface area) and were classified as light (<10%), medium (10% to 30%), and severe (>30%). Disease severity was further verified by oil red O staining of the aortic root (measured as averaged lesion per cross-section of the aortic sinus). The frequency of light, moderate and severe lesions in 12-month-old apolipoprotein E–/– mice on normal chow diet was 40%, 50%, and 10%, respectively. At 18 months, most apolipoprotein E–/– mice had severe lesions in their arteries.
Proteomic and Metabolomic Analysis
For proteomic and metabolomic analyses, aortas were rinsed thoroughly with cold phosphate-buffered saline to remove any blood components and frozen immediately in liquid nitrogen. Aortas from both sexes were used in all experiments. A detailed methodology is available online (http://atvb.ahajournals.org). Protocols can be downloaded from our website (www.vascular-proteomics.com).
Standard Methods
Western blotting, immunohistochemistry, and enzymatic assays are available online at http://atvb.ahajournals.org.
Statistical Analysis
Statistical analysis was performed using the analysis of variance and Student t test. Pairwise comparisons between metabolites were performed using the Bonferroni/Dunn test. Results were given as means±SE. P<0.05 was considered significant.
Results
Proteomic Analysis
To analyze changes in the proteome, we created protein profiles of aortas by 2-dimensional (2D) gel electrophoresis. Aortas were derived from 10-week-old and 12-month-old apolipoprotein E+/+ and apolipoprotein E–/– mice. Average gels were obtained from at least 4 animals per group. A direct overlay is presented in Figure 1. Using a broad range pH gradient (pH 3 to 10 NL), 2-D gels comprised 1500 protein features. Differentially expressed spots are highlighted in color (orange and blue indicate an increase in aortas of apolipoprotein E+/+ and apolipoprotein E–/– mice, respectively). Numbered spots were excised and subject to in-gel tryptic digestion. Protein identifications as obtained by mass spectrometry are listed in Table I (available online at http://atvb.ahajournals.org). For spots marked with an asterisk (*), further proof of identification was obtained by tandem mass spectrometry (n=38; Table II, available online at http://atvb.ahajournals.org). Representative spectra are shown online.
Figure 1. 2D map of proteins expressed in aortas of 10-week-old apolipoprotein E+/+ and apolipoprotein E–/– mice. Protein extracts were separated on a pH 3 to 10NL IPG strip, followed by a 12% SDS polyacrylamide gel. Spots were detected by silver staining. The figure represents a direct overlay of average gels from apolipoprotein E+/+ and apolipoprotein E–/– vessels. Average gels were created from 4 single gels (total n=8). Differentially expressed spots are highlighted in color (orange and blue for apolipoprotein E+/+ and apolipoprotein E–/– vessels, respectively). Proteins identified by mass spectrometry are marked with numbers and listed in Table I.
To quantitatively monitor protein changes during atherogenesis, aortas of 12-month-old apolipoprotein E–/– mice were classified according to their atherosclerotic surface area in vessels with light (<10%), medium (10% to 30%), and severe atherosclerosis (>30%). Confirmation was provided by oil red O staining (Figure 2A). Cholesterol levels were significantly increased in all subgroups of chow-fed apolipoprotein E–/– mice compared with wild-type controls (P<0.001 ANOVA), but no correlation was observed between cholesterol levels and disease severity in 12-month-old apolipoprotein E–/– mice (449±49 and 421±127 mg/dL in animals with light and severe lesions, respectively), which is in line with previous reports.10 Quantitative data on protein changes during disease progression are summarized in Table III (available online at http://atvb.ahajournals.org).
Figure 2. Atherosclerotic lesions in apolipoprotein E–/– mice. A, Representative photographs of oil red O-stained sections from aortic roots, indicating lesions (red color) of 10-week-old apolipoprotein E+/+ and apolipoprotein E–/– mice and 12-month-old apolipoprotein E–/– mice with light, medium, and severe disease. Original magnification x100. B and C, Western blots probed with antibodies to albumin, immunoglobulins, and apolipoprotein A1. Note that albumin undergoes extensive fragmentation in advanced lesions (C) and that immunoglobulins were barely detectable in apolipoprotein E+/+ aortas, but abundant in apolipoprotein E–/– vessels (B).
Accumulation of Serum Components
As expected, macrophage markers (MAC2, CapG) increased, whereas SMC markers (SM22) decreased, and serum proteins accumulated with lesion progression, including fibrinogen, transferrin, and hemopexin. Interestingly, immunoglobulin deposits were barely detectable in apolipoprotein E+/+ mice, but abundant even in aortas of young apolipoprotein E–/– mice (Figure 2B), forming 2 charge trains of molecular masses 25 700 and 50 800 Da with isoelectric point values of 7.8 to 5.8 on 2D gels. Whereas further immunoglobulin deposition occurred during lesion progression, albumin was subject to extensive fragmentation within advanced atherosclerotic plaques (Figure 2C). Apolipoprotein A1 (apoA1), the major protein fraction of high-density lipoprotein, whose protective anti-oxidative role in the cardiovascular system is well-established, was significantly reduced in aortas of apolipoprotein E–/– mice (Figure 2B).
Increased Oxidative Stress
Besides revealing differences in protein expression, 2D gel electrophoresis has the potential to display differences in posttranslational modifications. Redox-active cysteines constitute the main antioxidative component of peroxiredoxins.11 This protein family represents a special type of peroxidase as the protein is the reducing substrate itself; on oxidative stress, the cysteine in the active site is either oxidized to cysteine sulfenic acid or overoxidized to cysteine sulfinic acid. Whereas the first modification is DTT-sensitive and therefore undetectable in 2-D gels, the latter modification is DTT-resistant and results in a charge shift toward a more acidic isoelectric point.12 Thus, peroxiredoxins are often encountered as doublet spots in 2-D and the ratio of oxidized to reduced protein is a reliable surrogate marker for oxidative stress.11,12
1-Cys peroxiredoxin (1-Cys prx), a novel antioxidant conferring protection against oxidative membrane damage,13 was almost exclusively present as a reduced (basic) isoform in apolipoprotein E+/+ aortas, whereas oxidation of 1-Cys prx was detectable in vessels of young apolipoprotein E–/– mice, resulting in decreased abundance of the reduced isoform (Figure 3A). Consequently, the ratio of oxidized to reduced 1-Cys prx was 15-times higher in young apolipoprotein E–/– mice compared with wild-type controls (0.58±0.18 versus 0.04±0.03; P<0.05). Surprisingly, it temporarily normalized in vessels harboring light lesions (0.10±0.06 versus 0.04±0.03, nonsignificant); however, overall, there appeared to be a linear relationship between the extent of oxidation of 1-Cys prx and the extent of lesion formation in aortas of 12-month-old apolipoprotein E–/– mice (Figure 3A).
Figure 3. Oxidative stress in apolipoprotein E–/– aortas. The spot pair corresponding to 1-Cys peroxiredoxin (1-Cys prx) is marked with an arrow (A). Numbers correspond to protein identities in Table I. Quantitative changes in expression of the oxidized and reduced form of 1-Cys prx during atherogenesis are shown below. Note that 1-Cys prx is predominantly present as reduced protein in apolipoprotein E+/+ vessels but is oxidized in apolipoprotein E–/– vessels. Expression pattern of malic enzyme supernatant (MOD-1) (B). *Significant difference from wild-type controls, P<0.05, ** P<0.01.
Antioxidant Defense
1-Cys prx is able to reduce peroxidized membrane phospholipids by using glutathione (GSH) as a reductant.13 Under oxidative stress, GSH is oxidized to GSSG and subsequently reduced by GSH reductase through the coupling reaction of NADPH to NADP. Strikingly, GSH reductase activity was found to be increased in aortas of young apolipoprotein E–/– mice (78.4±12.4 IU/L versus 37.9±1.4 IU/L; n=3; P<0.05) and the oxidation state of 1-Cys prx in 12-month-old apolipoprotein E–/– mice correlated to the expression pattern of the cytosolic isoform of malic enzyme (MOD-1), which generates cytosolic NADPH,14 providing reducing equivalents for lipid synthesis and GSH recycling (Figure 3B). Aortas harboring only light lesions demonstrated a prominent change in MOD-1 (Figure 3B) associated with decreased oxidation of 1-Cys prx (Figure 3A), lower levels of the oxidative stress-induced protein HO-1 (Figure 4A and 4B), and a trend to higher GSH concentrations compared with age-matched vessels with advanced disease (42±0.9 versus 31±0.8 nmol/g wet weight; n=3; P=0.10). In contrast, upregulation of antioxidant proteins was only detectable in advanced, but not early, stages of disease (Figure 4A). This is consistent with previously published mRNA data, reporting decreased antioxidant transcription in aortas of apolipoprotein E–/– mice at the onset of lesion formation.15
Figure 4. Antioxidants in apolipoprotein E–/– aortas. Western blot analysis to determine expression levels of antioxidant proteins in aortic tissues, including heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), catalase 1, and peroxiredoxin 1 (A). Note that HO-1 expression is higher in aortas with advanced atherosclerosis compared with age-matched vessels harboring only light lesions (B).
Enzymatic Changes
Among the differentially expressed proteins were several glycolytic enzymes, including triose phosphate isomerase, transketolase, glyceraldeyde-3-phosphate dehydrogenase, enolase, and phosphoglycerate mutase, as well as all 3 subunits of the pyruvate dehydrogenase complex, which accomplishes the irreversible step from glycolysis to the trichloroacetic acid (TCA) cycle by converting pyruvate to acetyl-coenzyme A (CoA). Changes of enzymes involved in glucose metabolism were accompanied by a reduction of cytoplasmic malate dehydrogenase, which is involved in the transfer of cytosolic NADH into mitochondria. Concomitantly, short chain-specific acyl-CoA dehydrogenases, responsible for the degradation of short chain fatty acids to acetyl-CoA, were differentially expressed in aortas of young apolipoprotein E–/– mice and medium chain-specific acyl-CoA dehydrogenases became upregulated in vessels of old apolipoprotein E–/–mice (Table III).
Metabolite Changes
To clarify the metabolic net effect of these enzymatic changes, we measured vascular metabolites by high-resolution nuclear magnetic resonance spectroscopy. A representative proton magnetic resonance spectrum of an aortic extract is shown in Figure 5. Quantitative data are included as Table IV (available online at http://atvb.ahajournals.org), whereas Figure 6 shows a histogram displaying the relative metabolite ratios for apolipoprotein E–/– aortas derived from 10-week-old and 18-month-old apolipoprotein E–/– mice compared with wild-type controls. Decreased concentrations of alanine, a transamination product of pyruvate, were associated with a reduction of the adenosine nucleotide pool in aortas of young apolipoprotein E–/– mice and a coordinated but nonsignificant decline of other energy metabolites, such as total creatine and succinate, the oxidation of which is directly associated with respiratory chain reactions. The ratio of alanine to pyruvate was significantly decreased in young apolipoprotein E–/– mice compared with wild-type controls (1.7±0.8 versus 7.5±1.6; P=0.002) and remained reduced in old apolipoprotein E–/– mice (4.1±1.7 versus 7.5±1.6, P=0.019, respectively), but the adenosine nucleotide and creatine pool normalized. The metabolic profiles also revealed a significant increase in choline in aortas of old apolipoprotein E–/– mice. Interestingly, concentrations of trimethylamine oxide, a breakdown product of choline, were significantly higher in male than female aortas, suggesting a gender-specific difference in choline metabolism, which was independent of the apolipoprotein E genotype (inset in Figure 6). An additional comparison of metabolic profiles obtained from sex-matched aortas of 12-month-old apolipoprotein E–/– mice (n=3 in each group, 2 males, 1 female) revealed that aortas harboring only light lesions had a 1.7- and 1.9-fold increase in adenosine nucleotides (P=0.028) and succinate (P=0.060), respectively, but only half the glucose concentration (P=0.109) compared with aortas with severe disease.
Figure 5. Nuclear magnetic resonance spectra of a murine aorta derived from 18-month-old apolipoprotein E–/– mice. Within the aliphatic region (–0.05 to 4.2 parts per million) of the nuclear magnetic resonance spectra, resonances have been assigned to lactate (Lac), alanine (Ala), pyruvate (Pyr), acetate (Acet), succinate (Succ), carnitine (Car), choline (Cho), phosphocholine (PC), taurine (Tau), scyllo-inositol (Scy-ino), glycolic acid (Glyco), trimethylamine oxide (TMAO), glutamate (Glu), creatine (Cr), phosphocreatine (PCr). ADP+ATP and formate are showing in the aromatic region of the spectra (6.0 to 9.0 parts per million, see inset).
Figure 6. Comparison of metabolites in apolipoprotein E+/+ and apolipoprotein E–/– aortas. Relative changes of metabolites in aortas derived from 10-week-old (gray bars) and 18-month-old (black bars) apolipoprotein E–/– aortas compared with apolipoprotein E+/+ aortas (reference line). Abbreviations for metabolites are explained in the legend to Figure 5. Data are provided in Table IV. The inset highlights a gender difference for TMAO concentrations in murine aortas. *Significant difference with Bonferroni/Dunn, P<0.017.
Discussion
Our study provides evidence that immune activation, oxidative stress, and energetic impairment are among the earliest alterations in hyperlipidemic animals.
Inflammation
Immunoglobulin deposition within the vessel wall of apolipoprotein E–/– mice is close to peak levels even before lesion formation initiates and cannot be accounted for by impaired endothelial barrier function, because other serum components, such as albumin and fibrinogen, did not accumulate in vessels without overt atherosclerosis. In murine models, antibodies recognizing oxidized phospholipids correlate closely with lesion progression and regression16–18 and a class shift from IgG2a to IgG1, indicative for a switch of the T-cell response from Th1 to Th2, has been observed for circulating oxidized low-density lipoprotein antibodies in apolipoprotein E–/– mice.19 Similarly, we observed a preponderance of IgG1 within atherosclerotic lesions and mass spectrometry data obtained from the variable region of accumulated immunoglobulins suggest that at least some are directed against phosphocholine (gi30720232, anti-phosphocholine immunoglobulin heavy chain variable region [mus musculus], sequence coverage 33%). However, further studies will be required to allow for a more detailed characterization.
Oxidative Stress
Oxidative stress, the local imbalance between the ubiquitous formation of reactive oxygen species (ROS) and the equally ubiquitous antioxidant defenses, is thought to play an important role in vascular injury and atherogenesis.20–22 The complexity of the antioxidant defense have made it difficult to assess their impact on atherosclerosis as it is likely that knockout of individual ROS-generating or ROS-scavenging enzymes are compensated for by synergistic ones.23 Because the pathogenetic outcome is determined by the balance between pro-oxidants and antioxidants, measurements of individual enzymes at a single time are unlikely to shed much light and a more comprehensive approach is needed.23
Our proteomic data support the role of oxidative stress in atherogenesis: First, oxidation of 1-Cys prx, a reliable in vivo marker of oxidative stress,11,12 was significantly elevated in young apolipoprotein E–/– mice compared with wild-type controls. Second, the oxidation state of 1-Cys prx correlated to lesion size in aortas of 12-month-old mice indicating that reduced oxidative stress might attenuate lesion progression in apolipoprotein E–/– mice. Third, the observed reduction of oxidative stress in vessels with light lesions was not a result of increased expression of antioxidants, because protein levels of catalase 1, SOD-1, and peroxiredoxin 1 were similar to those in young apolipoprotein E–/– mice. The oxidation status of 1-Cys prx, however, showed a striking correlation to proteomic changes of malic enzyme supernatant (MOD-1). As demonstrated previously, such changes in the protein pattern are likely to reflect alterations in enzymatic activity.6–9 The soluble form of malic enzyme is 1 of 3 enzymes, apart from glucose 6-phosphate dehydrogenase and cytoplasmic isocitrate dehydrogenase, that can generate cytosolic NADPH,14 providing reducing equivalents for lipid synthesis, as well as for glutathione and thioredoxin reductase, which are of paramount importance in maintaining the reducing intracellular environment.24,25 The importance of thiol-based defense mechanisms in hyperlipidemia is supported by the increase in glutathione reductase activity in vessels of young apolipoprotein E–/– mice. This initial glutathione defense appears to be overwhelmed in advanced stages of disease as indicated by a rebound in the oxidation of 1-Cys prx, increased expression of HO-1, and lower glutathione levels compared with aortas harboring light lesions. Thus, upregulation of antioxidant proteins appears to be the last resort, once other counter-regulatory mechanisms cannot provide sufficient reducing equivalents to antagonize ROS, rather than the first attempt to confine oxidative stress. These findings would be consistent with previous studies reporting a weak glutathione-related enzymatic antioxidant shield in human atheroma.26
Despite a conjunct upregulation of antioxidant proteins in advanced stages of disease, deleterious consequences of reactive oxygen species became apparent, such as proteolysis of oxidatively damaged proteins.27 Albumin is degraded 50-times faster on oxidation,27,28 providing a possible explanation for its extensive fragmentation. Similarly, enzymes known to be susceptible to free radical-mediated inactivation such as aconitase and the Rieske protein of ubiquinol cytochrome C reductase, which contain iron-sulfur centers, a coordination complex with cysteine sulfurs of proteins, were altered in advanced stages of atherosclerosis.29,30 Such interactions are of potential importance, as damage to such complexes results in release of free iron and subsequent formation of hydroxyl radical, a highly reactive oxygen species, which perpetuates the vicious cycle of oxidative stress.31
Energy Metabolism
Metabolic disturbances are likely to be a key factor in both the initiation and progression of atherosclerosis. The upregulation of acyl-CoA dehydrogenases, the decrease in alanine, a transamination product of pyruvate, and the downregulation of cytoplasmic malate dehydrogenase, responsible for transferring cytosolic NADH produced during glycolysis into mitochondria,32 suggest that vascular cells might respond to hyperlipidemia by metabolizing lipids instead of glucose. Increased fatty acid oxidation would exert a negative feedback on the activity of the pyruvate dehydrogenase complex32 slowing down glucose metabolism, the main source of energy for the vasculature.33 Moreover, when excess fatty acids reach mitochondria, there is even a risk of uncoupling oxidation from phosphorylation with oxygen wastage.32 Insufficient phosphorylation of energy metabolites will cause their degradation and tissue depletion, providing a possible explanation for the observed reduction of the adenosine nucleotide pool in young apolipoprotein E–/– mice. Notably, breakdown products of adenosine are xanthine and hypoxanthine, both substrates for the xanthine oxidase, which is a powerful enzyme in the generation of ROS.34 It is noteworthy that the depletion of vascular energy metabolites coincided with increased oxidation of 1-Cys prx in young apolipoprotein E–/– mice, whereas attenuated lesion formation in 12-month-old apolipoprotein E –/– mice was associated with reduced oxidative stress and successful recovery of the adenosine nucleotide pool possibly serviced by increased glucose use. Supporting our findings are previous observations that insulin supplementation reduces lesion formation and oxidative stress in apolipoprotein E–/– mice35. In contrast, overexpression of the uncoupling protein 1 results in mitochondrial dysfunction and promotes atherosclerosis by depleting energy stores and increasing superoxide production.36 Thus, there is evidence that inefficient glucose and energy metabolism may contribute to oxidative stress and vascular disease in hyperlipidemic mice.
Study Limitation
A main obstacle for applying proteomic analysis to vascular pathology is the heterogeneous cellular composition of atherosclerotic plaques. Whereas smooth muscle cells dominate proteomic profiles of normal vessels, advanced lesions contain large numbers of monocyte-derived macrophages. Overall, the proteomic profiles were remarkably consistent in young and old apolipoprotein E–/– mice: only 2 macrophage proteins, namely CapG, which accounts for 0.6% of total macrophage proteins, and MAC-2, which is also abundant in activated macrophages, showed a significant increase in advanced stages of disease, indicating that the concentration of other macrophage proteins in aortic extracts was not high enough to allow detection on 2D gels. Thus, the proteomic and metabolic profiles remain dominated by vascular smooth muscle cells, facilitating data interpretation.
Finally, we should point out that our proteomic analysis revealed differential expression of several signaling proteins, but the vascular function of some proteins, eg, dihydropyrimidinase-like proteins 2 and 3, which are regulators of neuronal development and axonal outgrowth,37 is currently unknown. For others, there is evidence for their involvement in atherosclerosis: downregulation of Ras suppressor protein 1, an endogenous inhibitor of the Ras signaling pathway, during lesion progression, is consistent with a study reporting attenuation of lesion formation in apolipoprotein E–/– by inhibiting the Ras signaling pathway;38 the functional relevance of 14-3-3 gamma, an inhibitor of the protein kinase C signaling pathway, is supported by our findings that deficiency for protein kinase C delta accelerates neointima formation in a mouse model of vein graft arteriosclerosis.39
Summary
Our study is a first attempt to show how changes in the proteome and the metabolome are reciprocally connected during atherogenesis and provides evidence that attenuated lesion formation in apolipoprotein–/– mice is associated with reduced oxidative stress and successful recovery of the vascular energy pool.
Acknowledgments
The use of the facilities of the Medical Biomics Centre at St. George’s, University of London, is gratefully acknowledged.
This work was supported by grants from the British Heart Foundation and the Oak Foundation.
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