Molecular Analysis of Factors Contributing to Carotid Atherosclerotic Plaque Instability: Atheromatous versus Fibrous Plaques
Introduction: The molecular events in atherosclerotic carotid disease that trigger responses within the plaque resulting in plaque instability and thromboembolic cerebrovascular sequelae are unclear. Plaque instability may be related to apoptosis and extracellular matrix degradation. We compared atheromatous and fibrous plaques in terms of apoptosis, apoptosis-related proteins, cell cycle regulatory genes, death substrates for apoptosis, proteolytic activity and infiltrating cells. Methods:Apoptosis was detected in carotid endarterectomy plaques by the TUNEL assay. Immunohistochemical studies were performed to identify macrophages and infiltrating cells, localize the expression of bcl-2, bax, cyclin D-1, caspases, Fas, PARP, MMP-2 and MMP-9. Zymography was used to compare the proteolytic activity levels in the two types of plaques. Results:There were no inflammatory cells in the fibrous plaques. Apoptotic cell death was significantly higher (p<0.001) in atheromatous plaques as compared to fibrous plaques. Elevated levels of proteinases in atheromatous plaques were noted. There were significant increases in the expression of pro-apoptotic protein, bax (p<0.01), Fas (p<0.04), and CPP-32 (p<0.001) in atheromatous plaques as compared to fibrous plaques. Localization of bcl-2 in atheromatous as well as fibrous plaques was similar. There was differential expression of cell cycle protein, cyclin D-1. Conclusions:The greater amount of apoptosis and overexpression of mediators of apoptosis seen in atheromatous plaques suggest that deregulated apoptosis may be involved in the pathogenesis of unstable atherosclerotic plaques. Immunoreactivity to cytoplasmic death domain, Fas, and CPP-32, a prominent mediator of apoptosis, was consistent with the amount of apoptosis detected. Inflammatory cells and increased number of T-lymphocytes producing apoptotic stimuli could contribute to plaque instability. The increased levels of proteolytic activity in atheromatous plaques may make them vulnerable to rupture, and result in a cascade of thromboembolic cerebrovascular events.