Proteomic analysis of human vessels: Application to atherosclerotic plaques

Abstract

Atherosclerosis is a chronic disease that affects medium and large arteries. This process originates from the interaction between cells of the arterial wall, lipoproteins and inflammatory cells, leading to the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in acute clinical complications such as myocardial infarction and stroke. Owing to the heterogeneous cellular composition of the plaques, a proteomic analysis of the whole lesion is not appropriate. Therefore, we have studied the proteins secreted by human carotid atherosclerotic plaques, obtained by endarterectomy. Normal artery segments and different regions of the surgical pieces (noncomplicated plaque, complicated plaque with thrombus) were cultured in protein-free medium and the secreted proteins (supernatants) analyzed by two-dimensional gel electrophoresis. Normal artery segments secreted a moderate number of proteins (42 spots). However in the two-dimensional (2-D) gels (pH 3-10) of segments bearing a plaque, the number of spots increased markedly (154). The number of spots also increased (202) in the 2-D gels of artery segments with a ruptured plaque and thrombus. Thus, the more complicated the lesion, the higher the number of secreted proteins, suggesting the production of specific proteins relating to the complexity of the atherosclerotic lesion.

Role of Leukocyte Elastase in Preventing Cellular Re-Colonization of the Mural Thrombus

“…Using a method we have already proposed for the discovery of new secreted biological markers of atherosclerosis, 25 each layer was cut into small pieces (1 mm 3 ) and separately incubated in RPMI 1640 medium (Gibco, Cergy-Pontoise, France) for 24 hours at 37°C (2 ml/g of wet tissue). The conditioned media were then used for determination of secreted protease activities and for demonstrating the presence of solubilized fibronectin fragments.…”

Vascular proteomics

“…Using an ELISA which recognizes specifically the mature form of cathepsin D, we observed that cathepsin D levels on admission were significantly elevated compared to healthy subjects and stable patients, confirming the results obtained in monocytes. Interestingly, this protein was not detected by oligonucleotide array, but was clearly observed in the 2-DE gels, either from endartery samples [29][30][31] or circulating monocytes [135]. Interestingly, treatment with atorvastatin could modulate some of the altered proteins in the monocytes from ACS patients.…”

“…Although certain key risk factors have been identified, a highly sensitive and specific diagnostic assay that could provide information on the extent, growth, regression, stability, and type of atherosclerotic lesions remains to be identified. We have recently reported a novel approach for the study of atherosclerotic plaque secretome in the search of potential biomarkers of atherosclerosis [29][30][31][32]. Our strategy was to compare the secretome from normal and pathological arteries using a differential proteomic approach to identify new biological markers potentially released by the arterial wall into the plasma [33].…”

“…Incubation of endarterectomy samples versus control endarteries in a protein-free culture medium allowed us to harvest separately the proteins released from pathological and healthy areas. Using this approach, we have identified a number of proteins that are differentially released by normal or injured vessels [29,31] which belongs to different groups depending on their function, such as metabolic enzymes, structural proteins, protein transporters, transcription factors, antioxidants, and stress defense proteins. In particular, we have shown that in comparison with healthy mammary arteries, atherosclerotic plaques release less phosphorylated HSP27 [34], which was further confirmed by Western blot and ELISA.…”

“…3. Although several potential strategies are included in this figure only 2-DE has been used to date [29][30][31]. A priority in this workflow would be the definition of the true secretome, i.e., the identification of proteins which are actively synthesized and secreted by the normal and pathological arterial wall.…”