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Studies of differential gene expression in advanced carotid atherosclerosis

Final Activity Report Summary - CAROTID DISEASE (Studies of differential gene expression in advanced carotid atherosclerosis)

We studied a number of candidate biomarkers involved in atherosclerosis, its progression, ischemia, inflammation, plaque remodelling and angiogenesis. One of the main achievements of these studies was finding that C-reactive protein (CRP) deposited in the neointima is thought to be involved in the induction and promotion of pro-atherothrombosis phenotype in atherosclerotic lesions.

We have investigated the local expression of CRP and other pro-inflammatory biomarkers in human advanced carotid artery plaques. Carotid artery specimens from patients undergoing carotid endarterectomy for stenosis of greater than 70 per cent were grouped into one of three categories: ulcerated non-complicated plaques, fibrous plaques, or ulcerated complicated plaques exhibiting haemorrhagic transformation. Expression of CRP messenger RNA was much higher in ulcerated non-complicated plaques than in the other plaque types. Increased plaque CRP expression was unrelated to cardiovascular risk factors, treatment type, blood and serum parameters, and plasma CRP levels. Ulcerated non-complicated plaques also exhibited the highest expression of other genes involved in the inflammatory response (interleukin-6, cyclo-oxygenase 2 and monocyte chemotactic protein-1).

In ulcerated non-complicated plaques, immunostaining showed raised CRP levels around newly formed micro-vessels and in regions of high concentrations of inflammatory and endothelial cells. CRP immunostaining was absent in fibrous plaques and was weak or absent in ulcerated complicated plaques. We concluded that ulcerated non-complicated plaques appear to be 'biochemically active', which results in overexpression of CRP and other inflammatory markers. We suggest that patients are screened for these plaques.

Based on microarray screening of the carotid plaques we identified that normal cellular prion protein expression correlates with endoglin (CD105) positive micro-vessels in advanced carotid lesions. Normal cellular prion protein (PrPc) has multiple physiological functions; it is expressed by a variety of cells of the immune system and is present in endothelial cells. In the latter, PrPc is thought to participate in cell differentiation.

PrPc expression was examined using an Enzyme Immunometric Assay (EIA) in plasma samples from patients with advanced carotid disease. Carotid specimens obtained during endarterectomy and control normal-looking carotid arteries used for vascular transplants were studied for PrPc and CD105 (endoglin), a marker of active endothelial cells expression by Western blot and immunohistochemistry. TaqMan Low-Density Arrays were used to study PrPc and CD105 gene expression. We have demonstrated that PrPc is expressed in advanced carotid specimens and may be associated with the pro-angiogenic switch in these plaques.

Further, we started our work on CRP effects on differential gene expression in angiogenesis. This work for the first time demonstrated that CRP is pro-angiogenic and stimulates micro-vessel tube formation in vitro.