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Laser Capture Microdissection (LCM) for the Analysis of Macrophage Gene Expression from Atherosclerotic Lesions

serially-diluted standards and samples were analyzed by gel electrophoresis to confirm the presence of the specific amplicon (Fig. 3B). To show that LCM enriches macrophage-specific transcripts, RNA was extracted either from whole sections (analogous to homogenized tissue) or from LCM-derived CD68 immuno-positive macrophage foam cells. Real-time quantitative RT-PCR for CD68 was performed on equivalent amounts of RNA (100 pg) and the results normalized to the control gene, cyclophilin A. As shown in Fig. 3C, the LCM-derived RNA was significantly enriched in the mRNA for the macrophage specific marker, CD68, compared to whole section RNA (33.6-fold). To determine the potential cellular contamination of the laser captured cells by adjacent medial smooth muscle cells (SMC) either from non-specific tissue adherence to the thermoplastic film or from imprecise laser beam positioning, smooth muscle cellspecific α-actin was measured by real-time quantitative RT-PCR. As shown in Fig. 3D, in contrast to the level of α-actin in RNA from whole sections, in the LCM-derived RNA, α-actin expression was at background levels after 40 cycles of amplification. These results show that lesional macrophage foam cells can be precisely and selectively isolated from atherosclerotic vessels by CD68-guided LCM. To test whether RNA derived by LCM of macrophage foam cells can be utilized to quantitatively assess the transcriptional regulation of target genes implicated in atherosclerosis, laser captured lesional macrophage RNA from proximal aortas of LPS and control stimulated apolipoprotein E-/- mice were analyzed. Mice were administered either an intraperitoneal injection of the bacterial endotoxin lipopolysaccharide (LPS) (100 g) or vehicle only and, ~4 hours after treatment, the proximal aortas were processed for each mouse as described above. The relative gene transcript levels of the following genes were measured: vascular cell adhesion molecule-1 (VCAM-1), intercellular adh
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