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Laser capture microdissection for analysis of macrophage gene expression from atherosclerotic lesions
Jonathan E Feig1, Edward A Fisher
1The Marc and Ruti Bell Vascular Biology Disease Program, Department of Medicine (Cardiology), New York University School of Medicine, New York, NY, USA.
Insights
Researchers isolated RNA from macrophage foam cells in atherosclerotic plaques using laser capture microdissection. This technique revealed key gene expression changes during plaque regression, offering potential therapeutic targets for coronary artery disease.
Area of Science:
- Vascular Biology
- Atherosclerosis Research
- Molecular Medicine
Background:
- Coronary artery disease (CAD) due to atherosclerosis is a major global health issue.
- Previous studies homogenized atherosclerotic tissue, masking cell-specific gene expression.
- Macrophage foam cells are crucial in plaque progression, necessitating cell-specific analysis.
Purpose of the Study:
- To develop and apply a method for isolating foam cell-specific RNA from atherosclerotic plaques.
- To investigate gene expression changes in foam cells during atherosclerosis regression.
- To identify novel regulatory factors and therapeutic targets for CAD.
Main Methods:
- Laser capture microdissection (LCM) to isolate RNA from foam cells within plaques.
- Development of a transplantation-based mouse model (apoE-/-) to induce atherosclerosis regression by altering plasma lipid environment.
- Gene expression analysis of isolated foam cell RNA under regression conditions.
Main Results:
- Successful isolation of foam cell-specific RNA with significant transcript enrichment compared to whole tissue.
- Demonstrated rapid atherosclerosis regression in the mouse model, involving foam cell emigration to lymph nodes.
- Identified decreased inflammatory gene expression and increased cholesterol efflux gene expression in foam cells during regression.
- Found that chemokine receptor CCR7 expression is essential for plaque regression.
Conclusions:
- Laser capture microdissection (LCM) is a powerful tool for cell type-specific gene expression studies in atherosclerosis.
- Foam cell gene expression profiles change significantly during regression, highlighting potential therapeutic avenues.
- CCR7 plays a critical role in the regression of atherosclerotic plaques.
Abstract:
Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.
