Urine Proteome Analysis Reflects Atherosclerotic Disease in an ApoE~(-/-) Mouse Model and Allows the Discovery of New Candidate Biomarkers in Mouse and Human Atherosclerosis

摘要

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 electro-phoresis 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 alpha_1-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, alpha_1-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 1-antitrypsin fragments also significantly correlated with intraplaque collagen and alpha_1-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, alpha_1-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.