A Proteomic Focus on the Alterations Occurring at the Human Atherosclerotic Coronary Intima

Coronary heart disease remains the major cause of mortality in developed countries. In particular, coronary atherosclerosis is the responsible for the majority of the acute coronary syndromes. In the recent years, the understanding of atherosclerosis has experienced a drastic shift, because advances in basic research have pointed out the role of inflammation and the underlying cellular and molecular mechanisms that contribute to atherogenesis (1, 2). Intimal thickening produced by the migration of vascular smooth muscle cells (VSMCs)1 to the subendothelium, where they synthesize extracellular matrix (ECM), appears during normal development and aging (3), in response to minimal endothelium injury often produced by a disturbance in the pattern of blood flow at bending points and near bifurcations of the arterial tree (4). Atherosclerosis initiates at this locations with circulating leukocytes recruitment because of vascular endothelium alteration, which triggers the expression of adhesion molecules (VCAM) (3). Monocytes differentiate into macrophages within the arterial intima where they phagocyte lipids, finally turning into foam cells (5). Lipid accumulation within the intima leads to the formation of a central lipid core that is surrounded by a fibrous cap generated by migrated VSMCs (6). Proteomic analysis of plasma, circulating cells or atherome plaque from patients affected by atherosclerosis has lead to a better understanding of the initiation and development of this pathology, because proteins are the final effectors of all events triggered by lipid deposition onto the thickened intima. Although arterial tissue protein extracts have allowed characterizing many proteins involved in the pathogenesis of atherosclerosis (7), these studies have the limitation of measuring protein levels in all artery locations as a whole. Therefore, proteomic analysis of regions of interest isolated by laser microdisection (LMD) potentially gives more specific results. One limitation of this technique is that it may require long LMD times to recover sufficient amounts of protein. However, this limitation has been overcome by the combination of LMD with saturation labeling DIGE, a technique based on fluorescent labeling of proteins in Cys residues, which allows analyzing scarce samples by two-dimensional electrophoresis (2-DE) using less than 5 μg of total protein (8). In many cases, victims of acute coronary syndromes (ACS) do not present prior symptoms. For this reason, there is still need to develop novel early diagnosis biomarkers that could predict future cardiac events in asymptomatic patients. Because atherosclerosis initiates within the intima, we aimed to study atherosclerotic coronary intima proteome compared with nonatherosclerotic intima in the search for potential biomarkers of the disease right at the location where their expression levels start to increase/decrease. On the one hand, their discovery will lead to further understand the pathology and, as they may be finally released into plasma, they may constitute potential targets in early diagnosis, prognosis, or therapy. For this purpose, intima from human atherosclerotic coronary arteries and from preatherosclerotic coronaries and radial arteries was isolated by LMD. A differential abundance analysis was performed by saturation labeling DIGE resulting in the identification by MS of 13 proteins altered, 7 up-regulated and 6 down-regulated. Altered expression of a subset of these proteins was validated by immunohistochemistry (IHC) on an independent group of patients. A schematic view of the workflow of the study performed can be observed in Fig. 1. Fig. 1. Workflow of the differential protein abundance analysis of atherosclerotic coronary intima. A schematic view of the workflow from the study of atherosclerotic coronary intima differential proteome can be observed in this figure.