Molecular & Cellular Proteomics
Volume 11, Issue 7, July 2012, Pages M111.013847-1-M111.013847-13
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Research
Urine Proteome Analysis Reflects Atherosclerotic Disease in an ApoE−/− Mouse Model and Allows the Discovery of New Candidate Biomarkers in Mouse and Human Atherosclerosis*

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Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE−/− mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE−/− mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE−/− mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α1-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE−/− mice on HFD. Urinary excretion levels of collagen and α1-antitrypsin fragments also significantly correlated with intraplaque collagen and α1-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α1-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

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*

This work was supported in part by Deutsche Forschungsgemeinschaft Grant MU2727/3-1 (to C. v. z. M.), European Union Sixth Framework Programme InGenious HyperCare Grant LSHM-CT-2006-037093, SysKID Grant HEALTH-F2-2009-241544 (to E. S., P. Z., and H. M.), and a Future Fellowship from the Australian Research Council (to K. P.).

This article contains supplemental material.

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These authors contributed equally to this work.

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These authors contributed equally to this work.