title: Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model and allows the discovery of new candidate biomarkers in mouse and human atherosclerosis.
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.
authors.affiliation: Department of Cardiology & Angiology, University of Freiburg, Freiburg, 79104 Germany.
authors.forename: Constantin
authors.initials: C
authors.lastname: von zur Muhlen
authors.forename: Eric
authors.initials: E
authors.lastname: Schiffer
authors.forename: Christine
authors.initials: C
authors.lastname: Sackmann
authors.forename: Petra
authors.initials: P
authors.lastname: Zürbig
authors.forename: Irene
authors.initials: I
authors.lastname: Neudorfer
authors.forename: Andreas
authors.initials: A
authors.lastname: Zirlik
authors.forename: Nay
authors.initials: N
authors.lastname: Htun
authors.forename: Alexander
authors.initials: A
authors.lastname: Iphöfer
authors.forename: Lothar
authors.initials: L
authors.lastname: Jänsch
authors.forename: Harald
authors.initials: H
authors.lastname: Mischak
authors.forename: Christoph
authors.initials: C
authors.lastname: Bode
authors.forename: Yung C
authors.initials: YC
authors.lastname: Chen
authors.forename: Karlheinz
authors.initials: K
authors.lastname: Peter
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency* / Q000235:genetics; D050197:Atherosclerosis / Q000175:diagnosis / Q000209:etiology / Q000235:genetics / Q000652:urine*; D015415:Biomarkers / Q000652:urine; D024042:Collagen Type I / Q000652:urine*; D059305:Diet, High-Fat / Q000009:adverse effects; D018450:Disease Progression; D019075:Electrophoresis, Capillary; D004815:Epidermal Growth Factor / Q000652:urine*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D018345:Mice, Knockout; D010455:Peptides / Q000652:urine; D058226:Plaque, Atherosclerotic / Q000175:diagnosis / Q000209:etiology / Q000235:genetics / Q000652:urine*; D020543:Proteome / Q000378:metabolism; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein; D000515:alpha 1-Antitrypsin / Q000652:urine*
chemical_list: D001057:Apolipoproteins E; D015415:Biomarkers; D024042:Collagen Type I; D010455:Peptides; D020543:Proteome; D000515:alpha 1-Antitrypsin; D004815:Epidermal Growth Factor