title: 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.
authors.affiliation: Department of Medicine and The Zena and Michael A. Wiener Cardiovascular Institute, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Eugene
authors.initials: E
authors.lastname: Trogan
authors.forename: Robin P
authors.initials: RP
authors.lastname: Choudhury
authors.forename: Hayes M
authors.initials: HM
authors.lastname: Dansky
authors.forename: James X
authors.initials: JX
authors.lastname: Rong
authors.forename: Jan L
authors.initials: JL
authors.lastname: Breslow
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fisher
mesh_terms: D000199:Actins / Q000096:biosynthesis; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis; D001011:Aorta / Q000378:metabolism; D001057:Apolipoproteins E / Q000235:genetics / Q000502:physiology*; D001161:Arteriosclerosis / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000096:biosynthesis; D021984:Cyclophilin A / Q000096:biosynthesis; D004210:Dissection / Q000379:methods; D005487:Foam Cells / Q000502:physiology; D020869:Gene Expression Profiling / Q000379:methods*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D007834:Lasers; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure*; D051379:Mice; D008822:Mice, Transgenic; D009130:Muscle, Smooth / Q000166:cytology; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis; D000097382:CD68 Molecule
chemical_list: D000199:Actins; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D001057:Apolipoproteins E; D018932:Chemokine CCL2; D021984:Cyclophilin A; D018799:Intercellular Adhesion Molecule-1; D012313:RNA; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; C067980:CD68 antigen, human; D000097382:CD68 Molecule