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 CD68͞macrosialin by a rapid (Ϸ15-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, ␣-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 ␣-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 g 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.

Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading

“…The primer and probe sequences for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (GenBank accession no. M62766), Mac-2 (X16934), smooth muscle myosin heavy chain (NM 013607), calponin H1 (Z19542), and ␣-tropomyosin (NM 024427) are listed in Table 1, and those for CD68, ATP-binding cassette transporter 1 (ABCA1), ␣-actin, MCP-1, VCAM-1, and cyclophilin A (loading control for all marker genes), are the same as described (17,18). QRT-PCR standard curves (for each mRNA species) were constructed by using serial dilutions of mouse total RNA isolated from liver (HMG-CoA reductase), elicited peritoneal macrophages (CD68, Mac-2, and ABCA1), lesion-enriched aortic arch (MCP-1 and VCAM-1), or normal aorta (␣-actin, smooth muscle myosin heavy chain, ␣-tropomyosin, and calponin H1).…”

“…Total RNA was extracted by using a kit from Qiagen (Valencia, CA). The mRNA levels for specific genes were determined by QRT-PCR as described (17), using 10 ng of total RNA. The primer and probe sequences for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (GenBank accession no.…”

Gene expression changes in foam cells and the role of chemokine receptor CCR7 during atherosclerosis regression in ApoE-deficient mice

“…Serial sections (6 m thick) were obtained. For immunohistochemistry, sections were stained for CD68 (rat anti-mouse macrophages; Serotec; 2 g͞ml), as described (42). For ABCA1 (rabbit antihuman ABCA1; Novus Biochemicals; 10 g͞ml) or PPAR␥ (rabbit anti-mouse PPAR␥; Calbiochem; 1:100 dilution), sections were stained for 1 h at room temperature with the primary antibody, followed by incubation with biotinylated goat anti-rabbit Ig for 1 h, reaction with streptavidin-linked alkaline phosphatase, color development with substrate, and haematoxylin counterstaining.…”

Inhibition of Fibroblast Growth Factor Receptor Signaling Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice

“…Unfortunately, the antiphospho-FGFR antibody used in this study was not able to detect phospho-FGFR-positive cells within the time-frame required for adequate mRNA isolation from laser capture microdissected cells. 42 Development of higher affinity phospho-FGFR antibodies that bind rapidly to phosphoFGFRs will be required to address this issue.…”