Characterization of the Human Atheroma Plaque Secretome by Proteomic Analysis

Abstract

Atherosclerosis is one of the most common causes of death in developed countries. Atherosclerosis is an inflammatory process that results in the development of complex lesions or plaques that protrude into the arterial lumen. Plaque rupture and thrombosis result in the acute clinical complications of myocardial infarction and stroke. Although certain risk factors (dyslipidemias, diabetes, hypertension) and humoral markers of plaque vulnerability (C-reactive protein, interleukin-6, -10 and -18, CD-40L) have been identified, a highly sensitive and specific biomarker or protein profile, which could provide information on the stability/vulnerability of atherosclerotic lesions, remains to be identified. Recently, we have described a novel strategy consisting in the proteomic analysis of proteins released by normal and atherosclerotic arterial walls in culture. This method enables harvesting of proteins that are only secreted by pathological or normal arterial walls. By focusing only on the secreted proteins found in the tissue culture media, there is an intended bias toward those molecules that would have a higher probability of later being found in plasma. Using this approach, we have shown that carotid atherosclerotic plaques cultured in vitro are able to secrete proteins, and also that a differential pattern of protein secretion of normal arteries vs pathological ones has been observed. In this chapter, the proteomic analysis of the human atheroma plaque secretome is described.

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.…”

The pressure of finding human hypertension genes: new tools, old dilemmas

“…Several groups have reported on the application of proteomics techniques to analyse tissue or plaque specimens to study cardiovascular disease providing new insights into disease-related pathways. [45][46][47][48] Several studies have focused on the analysis of body fluids derived through minimally invasive means. These studies were either based on pooled blood specimens, 49,50 making individual sample classification impossible, or they included small patient cohorts without blinded studies for validation.…”