C. Felici

Proteomic analysis of atherosclerotic plaque.

Proteins play a fundamental role in the formation and progression of plaque, but proteomic analysis of plaque as a whole is difficult, due to its heterogeneous cellular composition and an abundance of plasma proteins. Several approaches to this problem are reported in the literature; they include proteomic analysis of vascular tissues, analysis of proteins released by normal and pathological arterial walls, proteomic analysis of vascular cells and proteomic analysis of blood. In a previous study, we proposed a new strategy for studying of proteome of plaque, which permits to select the proteins exclusive to plaque by the constructing of a reference synthetic gel. In the present work, we matched the spots of the reference synthetic gel with the spots of a pool of carotid plaque, in order to select only spots exclusive to plaque from the 2-dimensional electrophoresis of the pool of plaque. We selected some spots between those exclusive and identified them by mass spectrometry. Some proteins identified are involved in transport, others take part in elimination of toxic radicals, others are metabolic enzymes or structural proteins. This study represents an example of application of the new approach which we have proposed: the reference gel of proteome of plaque permits to select, on every sample of interest, only the spots exclusive to plaque; once selected, spots can be identified by mass spectrometry and, being typical of plaque composition, could represent novel markers of lesions and vascular risk.

Synthetic gel of carotid artery plaque.

Atherosclerosis is a complex disease that affects medium and large arteries, leading to the formation and progression of plaque. In this process the proteins play an essential role and as a consequence, proteomic-based strategies examining the protein content of cells or tissues could offer a useful approach for the study of plaque proteins. Due to the heterogeneous cell composition of plaque, proteome analysis of whole lesions is difficult, besides being also complicated by the presence of plasma proteins that cannot be completely eliminated. A good way to study variations in protein expression among series of gels is to construct a synthetic gel. This type of gel is obtained by averaging the positions, shapes and optical densities of spots in a given set of gels. To be included in the synthetic gel, spots must be found in at least three gels. To obtain a profile representative of the proteome of atherosclerotic plaque, canceling its high variability, we constructed a synthetic gel using an average of ten carotid plaque samples. We then compared it with an equivalent synthetic gel constructed using ten plasma samples from the same carotid surgery patients. For the comparison of two synthetic gels (plasma/plaque) we could discriminate plasma proteins from plaque proteins. Besides analysis of spots common to plasma, the synthetic gel is useful to detect spots exclusive to plaque, thus simplifying a very complex mixture.

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.