Abstract

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

Highlights

  • From the ‡Department of Cardiology & Angiology, University of Freiburg, Freiburg, 79104 Germany, ¶Mosaiques Diagnostics GmbH, Hannover, 30625 Germany, the ࿣Baker International Diabetes Institute Heart & Diabetes Institute, Melbourne, VIC 3004 Australia, the **Division of Cell and Immune Biology, Helmholtz Institute, Braunschweig, 38124 Germany, and ‡‡British Heart Foundation Glasgow Cardiovascular Research, University of Glasgow, Glasgow, G11 6NT United Kingdom

  • A systematic approach was undertaken to discover polypeptides that are specific for atherosclerosis

  • We focused on early forms of atherosclerosis and compared urine samples from week 7 either high fat diet (HFD) (n ϭ 11) or chow diet (CD) (n ϭ 12) with each other, resulting in 406 polypeptide discriminators

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Summary

Introduction

Several biomarkers have been described with the potential to reflect the extent of atherosclerosis and the risk of atherosclerosis-driven complications, including adhesion molecules such as VCAM-1 [5, 6], CD40L [7], and hsCRP [8] The definitions of these makers have proven to be helpful for the understanding of the pathomechanisms of atherosclerosis, its inflammatory component. In urine a proteome pattern could be established that reflected coronary artery disease in patients with an accuracy of 84%. Sequencing of these markers revealed a pattern consisting of different collagen fragments [11]. We provide evidence for the biological relevance of the defined marker proteins in human atherosclerosis

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