Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-deficient mice

Abstract

Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin by a rapid (approximately 15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, alpha-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of alpha-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 microg of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.

Figure 1

LCM of macrophage foam cells from atherosclerotic lesions of apoE-deficient mice. Selection of cells for laser capture was guided by immunohistochemical detection of macrophage specific marker CD68/macrosialin (red staining). (A) A proximal aortic lesion is shown before laser capture (200×; Inset: 30×). The CD68-positively stained cells are targeted for laser capture with a 15-μm laser diameter. Arrow in Inset denotes a representative lesion from which CD68-positive cells were isolated by LCM. (B) After the thermoplastic film is removed, the punched holes left after laser capture are seen in the remaining heterogeneous tissue. (C) The homogeneity of the captured material is confirmed under microscopic visualization before processing for RNA extraction. L, lumen.

Figure 2

Measurement of RNA transcripts by real-time quantitative RT-PCR. (A) Representative standard curves from real-time quantitative RT-PCR of CD68 (▴) and α-actin (■). The plot of cycle threshold versus log₁₀ of input starting mass of RNA shows a linear relationship for five orders of magnitude of starting RNA (the square of the correlation coefficients for CD68 and α-actin are 0.99 and 0.98, respectively). The mean is shown for each RNA mass from duplicate standards (the SD bars are not depicted because they are smaller than the figure symbol). (B) Electrophoretic analysis of the quantitative RT-PCR products. The products for CD68 (Upper) and α-actin (Lower) verify the specific amplification of appropriately sized amplicons (67 and 66 bp, respectively). The semiquantitative relationship (i.e., graded intensity of ethidium bromide stained bands) for the serial diluted standards (10 ng to 1 pg; lanes 1–5) corresponds to that measured in A. A water blank in place of the RNA was used as a negative control (lane 6).

Figure 3

LCM selectively enriches lesional macrophage RNA, as assessed by measurements of mRNA transcript levels of cell-specific markers. (A) Mean levels of macrophage-specific marker CD68 (relative to cyclophilin A) were assessed in LCM-derived lesional foam cell RNA (100 pg) and compared with that extracted from alternate whole sections. The levels of CD68 were markedly increased in the LCM sample (range: 0.035–0.037) compared with the whole-section RNA sample (range: 0.00096–0.0014). The 33.6-fold increase in CD68 levels in the LCM-procured sample attests to the enrichment of macrophage cell-derived RNA. (B) Selectivity of LCM as assessed by SMC α-actin mRNA content. The α-actin levels [mean (range): 0.0022 (0.0019–0.0025); normalized to cyclophilin A] in LCM-derived foam cells are compared with those in whole-section RNA. Although the levels of the control reference gene, cyclophilin A, were comparable between the two sample groups, there was no detectable amplification for α-actin in the LCM foam cell-derived RNA (the asterisk indicates where the data bar would have been).