Proteomic identification of biomarkers of vascular injury

Abstract

Predictive biomarkers may be beneficial for detecting, diagnosing, and assessing the risk of restenosis and vascular injury. We utilized proteomic profiling to identify protein markers in the blood following vascular injury, and corroborated the differential protein expression with immunological approaches. Rats underwent carotid artery injury, and plasma was collected after 2 or 5 weeks. Proteomic profiling was carried out by two-dimensional differential in-gel electrophoresis. The differentially expressed plasma proteins were identified by mass spectroscopy and confirmed by immunoblotting. Proteomic profiling by two-dimensional differential in-gel electrophoresis and mass spectroscopy revealed plasma proteins that were differentially expressed at 2 weeks after injury. Among the proteins identified included vitamin D binding protein (VDBP), aldolase A (aldo A), and apolipoproteinE (apoE). Immunoblotting results validated a significant reduction in these proteins in the plasma at 2 or 5 weeks after vascular injury, in comparison to control animals without vascular injury. These findings suggest that VDBP, aldo A, and apoE may be biomarkers for vascular injury, which will have important prognostic and diagnostic implications.

Keywords: Vascular injury; aldolase; angioplasty; apolipoprotein E; atherosclerosis; plasma marker; proteomic profiling; vitamin D binding protein.

Figure 1

Histological and immunohistochemical assessment of neointimal formation after vascular injury. Shown in panel A are histological cross sections of control carotid arteries without vascular injury. Shown in Panel B are carotid arteries at 2 weeks after injury, and Panel C are carotid arteries at 5 weeks after injury. Neointimal formation can be visualized by H&E staining at low and high magnification. Vascular cells could be visualized by CD31 staining for endothelial cells and α-SMA staining for smooth muscle cells. Arrows point to locations of neointimal formation. L denotes the location of the arterial lumen. Scale bar: 200 μm in the left-most panel and 100 μm elsewhere.

Figure 2

2D-DIGE showing differentially expressed proteins after 2 weeks of carotid artery injury in rats. Green spots represent proteins from the control plasma, whereas red spots indicate proteins from vascular injury samples. Proteins were separated according to molecular weight (MW) and isoelec-tric point (pI). Circles and numbers refer to spots in which proteins were identified by MS.

Figure 3

Identification of differentially expressed rat plasma proteins after 2 weeks of vascular injury. As shown in magnified view, the identified proteins included multiple isoforms of VDBP, apoE, and aldo A (P<0.05).

Figure 4

Validation of differentially expressed proteins by immunoblotting. (A) Representative im-munoblots for rodent plasma proteins. For the assessment of aldo A and apoE, plasma samples were resolved on an SDS-PAGE gel before immunoblotting. For VDBP, a native PAGE gel was run to preserve protein integrity of VDBP isoforms. Equal quantities of protein were loaded and confirmed by fibronectin. (B) Immunoblot data was quantified as a ratio of injury/control at each timepoint. * indicates statistically significant from control sample at the same time point (P<0.05). The horizontal line depicts the relative level of the control sample.