Abstract 47: Development and Use of a Novel Single-Cell Epigenetic Lineage Tracing Method to Identify Smooth Muscle--Derived Macrophage-Like Cells Within Human Atherosclerotic Lesions

Abstract

Aim: Smooth muscle cells (SMC) possess remarkable phenotypic plasticity that allows adaptation to changing environmental cues. Lack of definitive SMC lineage tracing studies and inability to identify phenotypically modulated SMCs within lesions due to loss of SMC marker gene expression raise major questions regarding the role of SMC in vivo in atherosclerosis progression. We hypothesize that a subset of cells within lesions that express macrophage markers are derived from SMC not monocytes and play a key role in determining plaque stability. Methods: We developed a novel lineage tracing based on detection of H3K4dime of the SM MHC gene, a SMC-specific epigenetic lineage marker we have previously shown is stable during phenotypic switching in vitro. Detection of H3K4dime of the SM MHC locus was done using a Proximity ligation assay (PLA) developed in our lab with an antibody targeting the biotinylated DNA probe for the SM MHC locus in conjunction with an anti-H3K4dime antibody. Use of secondary antibodies conjugated with oligonucleotides induces formation of circular DNA that serve as template for amplification, allowing visualization of co-localization of H3K4dime and the SM MHC gene (Duolink). Our new lineage tracing is suitable with human paraffin-embedded tissue sections (n=4) allowing investigation of SMC fate within human atherosclerotic lesion. Results: H3K4dime on the SM MHC gene (PLA+ cells) was found to be specific for SMCs and not found in any other cell types including adventitial fibroblasts, or endothelial cells. The method was validated using a SMC-specific lineage tracing mouse model wherein SM MHC Cre mice are crossed to ROSA flox STOP eYFP+/+ ApoE -/- mice. The H3K4dime/SM MHC PLA signal (i.e. PLA+) was exclusively found in eYFP+ cells. Moreover, some of the lesion SMCs were eYFP+/PLA+/SM α-actin-. Similarly, we identified PLA+ cells in human lesions that were positive for the macrophage marker CD68. Conclusion: Our new method permits definitive identification of SMC-derived cells within lesions even if they are not identifiable as SMC due to loss of SMC markers. Moreover, we provide exciting evidence that a significant fraction of macrophage-like cells in human lesions are derived from SMC. We postulate that transition of SMC to a macrophage state may be a key event leading to plaque destabilization and rupture with possible myocardial infarction or stroke.