With conventional molecular biological approaches, studies on proteins can only be conducted on a limited number of proteins. Advances in proteomic analysis now enable direct monitoring of global changes in protein expression and post-translational modifications, which will help identify new biomarkers for CVDs and potentially provide more insight into the treatment of CVDs [17
]. In particular, 2D-DIGE utilizes mass-and charge-matched spectrally resolvable fluorescent dyes (Cy3 and Cy5) to label two different protein samples in vitro
prior to two-dimensional electrophoresis (2DE). To date, 2DE/DIGE is still one of the central technologies in proteomics for the separation and differential comparison of thousands of proteins in a complex mixture [18
Since blood is easily accessible and harbors many proteins that remain to be fully characterized, it may be a valuable source for identifying diagnostic or prognostic markers of CVDs by proteomic profiling. Based on the 2D-DIGE results from the rat injury model, we identified a proteomic signature of blood proteins that may be useful as biomarkers of vascular injury. These proteins consisted of VDBP, aldo A, apoE, immunoglobulin lambda-2 chain C region and GFAP, and they were differentially expressed in high confidence intervals. Among them, VDBP, aldo A and apoE were potential biomarkers for vascular injury.
One of the main findings of this study was that VDBP protein levels change in the presence of vascular injury. VDBP is a member of the albumin superfamily of binding proteins and exists in 3 dominant full-length isoforms, in addition to over 120 genetic variants [25
]. Besides serving as a transporter of vitamin D, VDBP also plays an important role in response to tissue injury, in which VDBP can be converted to a macrophage-activating factor (VDBP-MAF) to stimulate macrophages [26
]. Another role of VDBP in response to injury is to scavenge for vascular and extracellular actin as a result of cellular necrosis, and VDBP has been shown to be in lower circulating concentrations in the presence of inflammatory or necrotic diseases [25
]. Furthermore, VDBP is also implicated in a numerous diseases, including chronic obstructive pulmonary disease, cancer, and trauma [29
]. Using proteomic profiling, we identified 4 isoforms of VDBP, of which included isoforms with significant induction or reduction at 2 weeks after vascular injury in rat plasma. The differential regulation of VDBP isoforms suggests that there may be a unique VDBP expression pattern that characterizes the progression of vascular injury and restenosis. Immunoblot-ting data showed an overall reduction of VDBP expression in the plasma of injured rats, and this trend of reduced VDBP expression remained consistent at both 2 weeks and 5 weeks after vascular injury. However, further studies are necessary to elucidate the mechanism by which VDBP is regulated in the development of vascular injury.
ApoE is an important ligand for receptor-mediated uptake of triglyceride-rich lipoproteins and mediator for extracellular lipoprotein ho-meostasis [32
]. Its function in the low density lipoprotein receptor-related protein (LRP)-mediated pathway includes binding to the lipoprotein for delivery to the receptor, binding to heparin sulfate proteoglycans to mediate ligand -receptor interactions, and interacting with the LRP receptor to regulate uptake of the lipoprotein [33
]. ApoE deficiency or defect is known to be associated with hyperlipidemia [34
], atherosclerosis [35
], and Alzheimer's disease [36
]. In accordance with previous reports [37
], significant apoE reduction could be detected in rat plasma after 5 weeks of vascular injury, concomitant with neointimal thickening. These results suggest that apoE reduction may be associated with vascular injury, but further research would be required to explore this possibility.
Another metabolism-related marker identified by proteomic profiling was aldo A. Aldo A is 1 of 3 isoforms of aldolase, whose functional form is a homo or hetero-tetramer [38
]. Aldo A plays an important role in muscle-specific glycolysis by catalyzing the cleavage of fructose-1, 6-bisphosphate into glyceraldehyde-3-phosphate and dihydroacetate phosphate [39
]. Although the role of aldo A in diseases is largely unknown, the data from the rodent vascular injury study suggests that aldo A reduction is evident in the plasma at 2 weeks after injury and implicates aldo A as a biomarker of vascular injury.
Previous studies have shown evidence of plasma proteins that are associated with vascular injury, including interleukin-18, thrombo-modulin, and soluble CD39 [40
]. These proteins, when combined with the ones identified by our proteomic analysis, may serve as a robust biomarker panel that is predictive of vascular injury.
Although the rodent model of vascular injury is limited by the absence of cardiovascular risk factors, it distinguishes the proteomic changes associated with injury from those resulting from other disease factors. This model provides initial clues to unravel the complex events that underlie proteomic changes in response to pathophysiological events. Further studies using established disease models are warranted and will further elucidate the roles of VDBP, aldo A, and apoE in vascular disease in the presence of cardiovascular risk factors. In addition, future clinical studies may provide further validation of these biomarkers.
In summary, we utilized high-throughput proteomic profiling and traditional immunological approaches and identified VDBP, aldo A, and apoE levels to be differentially expressed in an experimental model of rat carotid injury. These finding have important prognostic and diagnostic implications in the treatment of vascular injury.