Urine proteome analysis reflects atherosclerotic disease in an ApoE-/- mouse model

Non-invasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, our recent work has yielded a biomarker pattern in humans reflecting coronary artery disease with high accuracy. In order to allow characterization of the pattern's pathophysiological roles in atherogenesis and to identify novel urinary polypeptide patterns reflecting early stages of atherosclerosis, we analyzed urine of mice. ApoE-/- mice develop atherosclerosis when fed a high cholestrol diet.
We collected urine of ApoE-/- mice either on high fat diet  or chow diet  over 12 weeks and analyzed it with capillary electrophoresis coupled to mass spectrometry. We were able to identify 16 atherosclerosis-specific polypeptides, which allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression in a blinded test set. Sequencing of the discovered polypeptides identified fragments of α1-antitrypsin, EGF, kidney androgen regulated protein (KAP) and collagen. Using immunohistochemistry, α1-antitrypsin, EGF and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE-/- mice. 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, presence of collagen type I, α1-antitrypsin, and EGF was also confirmed in human atherosclerotic disease.
Our work in mice exemplifies the potential of a novel multimarker approach for the non-invasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this example highlights the possibilities to dicover novel biomarkers relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.