Feig Jonathan E, Fisher Edward A
The Marc and Ruti Bell Vascular Biology Disease Program, Department of Medicine (Cardiology), New York University School of Medicine, New York, NY, USA.
Methods Mol Biol. 2013;1027:123-35. doi: 10.1007/978-1-60327-369-5_5.
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.
冠状动脉疾病由动脉粥样硬化引起，是西方世界的主要死因。此前大多数研究对动脉粥样硬化动脉（一种细胞组成混合的组织）进行匀浆处理，以确定斑块形成中的因素，从而模糊了与特定细胞类型相关的信息。由于巨噬细胞泡沫细胞是动脉粥样硬化斑块进展的关键介质，我们推断对这些细胞进行基因分析将为新的调节因子和潜在治疗靶点提供具体见解。我们在血管生物学领域首次证明，可通过对斑块进行激光捕获显微切割（LCM）来分离泡沫细胞特异性RNA。正如预期的那样，与全组织相比，观察到泡沫细胞特异性RNA转录本有显著富集。此外，由于动脉粥样硬化的消退是一个诱人的临床目标，我们开发并报道了一种基于移植的小鼠模型。这包括让载脂蛋白E基因敲除（apoE-/-）小鼠形成斑块，然后将斑块的血浆环境从高脂血症转变为正常血脂血症。在这些条件下，斑块迅速消退，这一过程涉及斑块泡沫细胞迁移至局部和全身淋巴结。使用LCM，我们能够证明在消退条件下，炎症基因在泡沫细胞中的表达降低，但胆固醇流出基因上调。有趣的是，我们还发现趋化因子受体CCR7（一种已知的树突状细胞迁移因子）表达增加是消退所必需的。总之，本章所述的LCM方法已经带来了许多惊人的发现，可能会进一步促进对动脉粥样硬化各个阶段以及基因、药物和环境扰动后动物和人类斑块中细胞类型特异性基因表达的研究。
