( Dr. Eugene Trogan Ph.D. Mount S...)Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions

Dr. Eugene Trogan, Ph.D., Mount Sinai School of Medicine of New York University

Abstract
Macrophage foam cells are integral in the development of atherosclerotic lesions, however gene expression studies are complicated by the cellular heterogeneity of atherosclerotic plaque. This application note describes a protocol for LCM of cells identified immunohistochemically, followed by real-time RT-PCR to selectively analyze RNA from foam cells of apolipoprotein E-deficient mice. The specificity of the procedure and the measurement of gene induction in laser captured cell populations after an in vivo perturbation are illustrated. These techniques will facilitate the study of atherosclerosis.

Introduction
Macrophage foam cells are critical in the development of atherosclerosis (1-3). Therefore, a better understanding of the gene expression changes in foam cells during disease progression and regression has become an important goal in order to develop potential therapies and interventions (4-6). However, the study of macrophage foam cell gene expression in arterial lesions is hampered by the cellular heterogeneity of arterial tissue, which, besides macrophages, also contains lymphocytes, smooth muscle cells, endothelial cells, and adventitial fibroblasts. To overcome these technical obstacles, we describe here a method for the use of LCM (7, 8) to selectively procure macrophage foam cells from arterial lesions (identified by the macrophage-specific marker, CD68/ Macrosialin (9, 10). RNA extracted from the laser captured foam cell material was used to quantify, by real-time quantitative RT-PCR, the fold of enrichment and to measure the induction of specific transcripts in response to an inflammatory stimulus. These methods make possible the quantitative analysis of gene expression in macrophage foam cells and add a powerful dimension to the study of atherosclerosis.

Equipment and Reagents
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