Carotid atherosclerotic plaques: proteomics study after a low-abundance protein enrichment step.

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin ß-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

CD36 mRNA and Protein Expression Levels Are Significantly Increased in the Heart and Testis of apoE Deficient Mice in Comparison to Wild Type (C57BL/6).

CD36, an 88kd-adhesion molecule, plays a major role as a scavenging receptor implicated in cellular lipid metabolism. Secretory mammary epithelium, microvasculature endothelium, adipocytes, smooth muscle cells, and platelets express CD36. In addition, CD36 expression is significantly enhanced in macrophages differentiating into foam cells. The effect of pathological levels of cholesterol, as observed in apoE(-/-), on vascular CD36 expression is, at this stage, not known. In this study, a quantitative analysis of CD36 transcription and protein expression levels, present in tissues of male C57BL/6 and apolipoprotein-E (apoE) deficient mice was carried out by Northern and Western blots. Four-week-old animals were fed a chow diet over different periods of time (0, 6, 16, or 20 weeks). Immunohistochemistry was used to localize CD36 protein expression in the heart and testis. Results indicate that CD36 transcription is increased in hearts of apoE deficient animals (100% higher at 6 weeks, and 30% higher at 16 and 20 weeks) in comparison to wild type. This was confirmed at the protein level, which showed an increase of at least 100% at 6 weeks, and between 40% to 50% increase at 16 and 20 weeks of apoE(-/-) mice compared to controls. In addition, CD36 transcription levels were significantly increased in testis of apoE animals (at least 100% at 6, 16, and 20 weeks) compared to C57BL/6 wild type. Such an increase was also confirmed at the protein level (65% increase at 16 weeks in apoE mice compared to control). Finally, localization of CD36 protein expression by immunohistochemistry showed that it was expressed in the capillaries of heart and testis endothelial cells and also at the head of spermatozoid during spermatogenesis. These results indicate that high circulating cholesterol levels, in apoE deficient mice, significantly enhance the expression of CD36 in the heart and testis. Such enhanced CD36 expression might lead to organ remodeling and/or dysfunction.

Local carotid atherosclerotic plaque proteins for the identification of circulating biomarkers in coronary patients.

OBJECTIVE: To identify circulating biomarkers that originate from atherosclerotic vulnerable plaques and that could predict future cardiovascular events. METHODS: After a protein enrichment step (combinatorial peptide ligand library approach), we performed a two-dimensional electrophoresis comparative analysis on human carotid plaque protein extracts (fibrotic and hemorrhagic atherosclerotic plaques). In silico analysis of the biological processes was applied on proteomic data. Luminex xMAP assays were used to quantify inflammatory components in carotid plaques. The systemic quantification of proteins originating from vulnerable plaques in blood samples from patients with stable and unstable coronary disease was evaluated. RESULTS: A total of 118 proteins are differentially expressed in fibrotic and hemorrhagic plaques, and allowed the identification of three biological processes related to atherosclerosis (platelet degranulation, vascular autophagy and negative regulation of fibrinolysis). The multiplex assays revealed an increasing expression of VEGF, IL-6, IL-8, IP-10 and RANTES in hemorrhagic as compared to fibrotic plaques (p<0.05). Measurement of protein expressions in plasmas from patients with stable and unstable coronary disease identified a combination of biomarkers, including proteins of the smooth muscle cell integrity (Calponin-1), oxidative stress (DJ-1) and inflammation (IL-8), that allows the accurate classification of patients at risk (p=0.0006). CONCLUSION: Using tissue protein enrichment technology, we validated proteins that are differentially expressed in hemorrhagic plaques as potential circulating biomarkers of coronary patients. Combinations of such circulating biomarkers could be used to stratify coronary patients.

CD36 inhibitors reduce postprandial hypertriglyceridemia and protect against diabetic dyslipidemia and atherosclerosis.

CD36 is recognized as a lipid and fatty acid receptor and plays an important role in the metabolic syndrome and associated cardiac events. The pleiotropic activity and the multiple molecular associations of this scavenger receptor with membrane associated molecules in different cells and tissues have however questioned its potential as a therapeutic target. The present study shows that it is possible to identify low molecular weight chemicals that can block the CD36 binding and uptake functions. These inhibitors were able to reduce arterial lipid deposition, fatty acid intestinal transit, plasma concentration of triglycerides and glucose, to improve insulin sensitivity, glucose tolerance and to reduce the plasma concentration of HbAc1 in different and independent rodent models. Correlation between the anti-CD36 activity of these inhibitors and the known pathophysiological activity of this scavenger receptor in the development of atherosclerosis and diabetes were observed at pharmacological doses. Thus, CD36 might represent an attractive therapeutic target.

The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidized low-density lipoprotein.

CD36, a major adhesion molecule expressed by monocytes/macrophages, plays a key role in the binding and internalization of oxidized low-density lipoprotein (OxLDL). This adhesion molecule, a member of an important scavenger receptor family, contains a very short C-terminal cytoplasmic tail that is known to induce intracellular signalling events. However, the domains on the cytoplasmic tail involved in such signal transduction are unknown. In this study, we have investigated the functional components of the cytoplasmic tail by site-directed mutagenesis coupled with functional OxLDL and monoclonal antibody (mAb) binding studies. Seven truncated or punctual CD36 constructs, localized in the cytoplasmic tail, were produced by site-directed mutagenesis. Each construct was stably expressed in HEK293 cells. We used a quantitative and a qualitative method, labelling OxLDL with either iodine or rhodamine, to determine the functional importance of the cytoplasmic domains in OxLDL internalization. Results indicate that: (1) a deletion of the last amino-acid (construct K472STOP) significantly reduces, compared with wild-type, the binding, internalization and degradation of OxLDL; (2) truncation of the last six amino-acids (construct R467STOP) significantly reduces OxLDL binding; (3) the above two constructs (K472STOP and R467STOP) showed a reduced rate of OxLDL internalization compared with wild-type; (4) the binding and rate of internalization of an anti-CD36 monoclonal antibody (10/5) was not affected by the above mentioned mutants (K472STOP and R467STOP), compared with wild-type. This study shows, for the first time, a specific site on the CD36 cytoplasmic tail that is critical for the binding, endocytosis and targeting of OxLDL.