O231	The oxidative phosphorylation pathway is differentially regulated between macrophages microdissected from early and advanced atherosclerotic lesions.
1N.Kisters, 1M.Faessen, 2J.O.Fledderus, 2O.L.Volger, 1K.B.Cleutjens, 1M.J.Daemen, 1A.P.Bijnens
1Department of Pathology, CARIM, University of Maastricht, Maastricht, NL; 2Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, NL.

Most gene expression studies in atherosclerosis use RNA from cell cultures or from entire atherosclerotic lesions. These studies are limited because cultured cells do not represent the full spectrum of cell interactions, while cellular heterogeneity of entire atherosclerotic specimens makes gene expression profiles difficult to interpret. Therefore we used laser capture microdissection (LCM) to isolate macrophages from early and advanced human atherosclerotic lesions and compared the generated microarray derived gene expression profiles. We hypothesize that the gene expression profile differs between macrophages from early and advanced human atherosclerotic lesions. Carotid arteries from males older than 50 years were obtained from autopsy and immediately imbedded in Tissue TekTM. Lesions were classified as early (n=4; 59±11 years) or advanced lesions (n=4; 60 ±14 years). From each lesion, 500 – 2000 macrophages were isolated by LCM (Arcturus Pixcell II) from serial frozen sections using CD68 immuno-positivity on parallel sections as a navigator. The isolated RNA was subjected to 3 rounds of T7 polymerase based amplification. The obtained cRNA of individual lesions was hybridized in duplicate to custom-made 65 mer oligonucleotide arrays (Sigma-Genosys/Compugen library) against a common reference sample. Analysis of differential gene expression was performed on LOESS normalized expression data. Out of 18.658 genes, 132 genes showed a significant (P<0.05) over 1.4 fold modulated expression between macrophages from early and advanced lesions (84 up- and 48 down regulated genes in macrophages of advanced lesions). Bioinformatic analysis revealed that genes involved in inflammation (e.g. RANTES, MCP-2) and lipid metabolism (e.g. ABCA-1) were not differentially expressed. Interestingly, differential expression of 5 genes involved in the oxidative phosphorylation (e.g. Cox 5A) suggested a metabolic switch in macrophages during lesion progression. In conclusion, with laser capture microdissection we identified the oxidative phosphorylation pathway to be differentially regulated in macrophages isolated from early and advanced atherosclerotic lesions.

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