Identification of Phenotypically Modulated SMCs within Atherosclerotic Lesions and the Role of Pluripotency Factors, KLF4 and OCT4, in their Modulation 603 views

Cardiovascular disease is the leading cause of death in developed countries, and there is compelling evidence that the majority of these fatalities are secondary to rupture of unstable atherosclerotic plaques. However, the mechanisms that control plaque stability are poorly understood, although it is widely believed that plaques having a decreased ratio of cells positive for smooth muscle cell (SMCs) markers such as ACTA2 relative to macrophage markers are more likely to rupture with consequent clinical sequelae. While several studies have shown that SMCs can undergo phenotypic modulation into an inflammatory or macrophage-like state in vitro, there is no direct evidence this occurs in vivo or that the process is functionally important in the pathogenesis of atherosclerosis. Studies within this dissertation are focused on identifying phenotypically modulated SMCs within atherosclerotic lesions using a newly developed conditional SMC specific lineage tracing mouse model, and a novel method that allows for the visualization of histone modifications of single genomic loci with single-cell resolution in formaldehyde-fixed paraffin-embedded tissue sections.Results show that dimethylation of lysine 4 of histone H3 (H3K4me2) at the MYH11 locus is restricted to the smooth muscle cell (SMC) lineage in human and mouse tissue sections and that the mark persists even in phenotypically modulated SMC in atherosclerotic lesions that show no detectable expression of SMC marker genes. We also show that traditional methods for staining and detecting SMCs are unable to detect ≥80% of SMC-derived cells within advanced atherosclerotic lesions of Apoe-/- mice following 18 weeks of Western diet feeding. Remarkably, nearly a quarter of macrophages identified by markers such as LGALS3 are of SMC- and not myeloid origin as has been assumed in previous studies in the field. Moreover, using our novel single cell epigenetic assay we provide evidence that a population of SMC-derived cells within advanced human coronary artery atherosclerotic lesions also express the macrophage marker CD68. We also demonstrate that pluripotency factors krϋppel-like factor 4 (Klf4) and octamer binding factor 4 (Oct4) play critical, albeit distinct roles in regulating SMC phenotypic switching. SMC specific knock out (KO) of Oct4 in Apoe-/- mice fed a high fat Western diet for 18 weeks resulted in a significant reduction in phenotypically modulated SMCs within atherosclerotic lesions but resulted in lesions that were larger but less stable. In contrast, SMC specific knock out (KO) of Klf4 in Apoe-/- mice fed a high fat Western diet for 18 weeks showed no significant change in the number of phenotypically modulated SMC within the plaque but was associated with decreased lesion size and multiple changes consistent with increased plaque stability. Taken together, results demonstrate that the contribution of SMCs to atherosclerotic lesion size and stability has been greatly underestimated by previous studies in the field. Moreover, studies are the first to provide direct evidence that changes in SMC phenotypic switching can exert dominant effects on the size, cellular composition, and stability of advanced atherosclerotic plaques, and indicate that regulating SMC phenotypic states should be investigated as a potential therapeutic target to help stabilize atherosclerotic plaques to reduce possible clinical sequelae including plaque rupture, myocardial infarction, and/or stroke.

PHD (Doctor of Philosophy)

atherosclerosis; smooth muscle cells; macrophages; plaque stability

English

All rights reserved (no additional license for public reuse)

2014-04-15