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Heterogeneity of gene expression in human atheroma unmasked using cDNA representational difference analysis

Department of Medicine, Division of Cardiovascular Medicine, Addenbrooke’s Centre for Clinical Investigation, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom

The rupture of an atherosclerotic plaque can have profound consequences, such as myocardial or cerebrovascular infarction. The complex interactions of vascular smooth muscle cells (VSMCs) with inflammatory and immune cells are thought to contribute to both plaque genesis and stability. Key to our understanding of these processes is the identification of genes expressed in human atheromatous lesions. We have employed cDNA representational difference analysis (RDA) to investigate the differences in gene expression between normal and atherosclerotic human vessels. Thirty-one cDNA clones representing sequences expressed in atheroma were isolated, many of which encoded components of inflammatory and immune pathways. The reciprocal experiment, to identify genes expressed in the healthy vasculature, identified two genes associated with the contractile functions of VSMCs. Semiquantitative RT-PCR analysis of expression of these genes in forty samples, derived from healthy and atheromatous vessels, demonstrated marked heterogeneity of gene expression between lesions, although several of the genes were preferentially expressed in atherosclerotic lesions. In situ hybridization identified subsets of macrophages at sites of neovascularization within the lesion and intimal VSMCs as expressing the disease-associated genes. In conclusion, cDNA RDA is a useful, fast, and efficient technique for studying differential gene expression particularly when clinical material is limiting.

atherosclerosis; inflammation; smooth muscle; macrophages

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