Proteomic and Metabolomic Analyses of Atherosclerotic Vessels From Apolipoprotein E-Deficient Mice Reveal Alterations in Inflammation, Oxidative Stress, and Energy Metabolism

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 Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ 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 Ϫ/Ϫ 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. Key Words: animal model Ⅲ apolipoprotein E Ⅲ atherosclerosis Ⅲ metabolomics Ⅲ oxidative stress Ⅲ proteomics T he generation of apolipoprotein E-deficient (apolipoprotein E Ϫ/Ϫ ) mice 1,2 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 insufficient 5 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 vivo 7,8 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 t...

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice

“…Many proteins, acknowledged as characteristic of vascular disease progression, are identified in this animal study as strongly linked to nutritional B vitamin depletion (noted in parentheses). Medial degeneration in the human ascending aorta alters the expression of proteins critical in vascular remodelling, cytoskeleton organisation and muscle contraction (actin, collagen, profilin, myosin, vimentin), VSMC phenotype (transgelins), protein folding and ROS detoxification (Hsps, peroxiredoxin, protein disulphide isomerase), and iron transport and inflammation (serotransferrin) (Mayr et al 2005). The HUPO Plasma Proteome Project identified more than 300 human plasma proteins as strong biomarkers of vascular function and/or disease.…”

“…Protein profiling has also been employed to investigate mechanism of vascular dysfunction in animal models. Serotransferrin, 78 kDa glucose-regulated protein and pyruvate kinase isozyme M1/M2 are elevated in experimentally induced rat heart failure (Ingwall and Weiss 2004), while fibrinogen, collagen, peroxidase and transgelin are overexpressed in atherosclerotic mice (Mayr et al 2005;Wu et al 2007). Transgelin-1 is increased in the artery wall of hypertensive rats (Delbosc et al 2008).…”

“…Moreover, they have proven to be powerful tools in rodents studies for providing mechanistic insight into how vascular disease progresses and is influenced by environmental factors such as diet (Mayr et al 2005;Almofti et al 2006).…”

Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders. Proteomics approaches to the systems biology of cardiovascular diseases

“…One cell type may compose the bulk of tissue, as in myocardium or liver, but in pathologic settings significant numbers of inflammatory cells may be present or there may be a change in the normal proportion of parenchymal to stromal cells (e.g., cardiomyocytes to fibroblasts). The analysis of atherosclerotic lesion material is perhaps an extreme example of this (22). The use of isolated or cultured cells addresses the complex cell type issue but raises other concerns, such as relevance to the in vivo setting.…”

Vascular proteomics

“…The complementarities in the identification of novel biomarkers using different approaches are highlighted in the lower part of the figure. [23]. An average of 1500 spots were visualized in the 2-DE of aorta samples, from which 79 proteins were found to be altered during the different stages of atherogenesis.…”