Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.

Hepatocyte-like cells (HLCs) are derived from human pluripotent stem cells (hPSCs) in vitro, but differentiation protocols commonly give rise to a heterogeneous mixture of cells. This variability confounds the evaluation of in vitro functional assays performed using HLCs. Increased differentiation efficiency and more accurate approximation of the in vivo hepatocyte gene expression profile would improve the utility of hPSCs. Towards this goal, we demonstrate the purification of a subpopulation of functional HLCs using the hepatocyte surface marker asialoglycoprotein receptor 1 (ASGR1). We analyzed the expression profile of ASGR1-positive cells by microarray, and tested their ability to perform mature hepatocyte functions (albumin and urea secretion, cytochrome activity). By these measures, ASGR1-positive HLCs are enriched for the gene expression profile and functional characteristics of primary hepatocytes compared with unsorted HLCs. We have demonstrated that ASGR1-positive sorting isolates a functional subpopulation of HLCs from among the heterogeneous cellular population produced by directed differentiation.

Myocardial Infarction-Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries.

OBJECTIVE: Coronary artery disease (CAD), including myocardial infarction (MI), is the main cause of death in the world. Genome-wide association studies have identified dozens of single nucleotide polymorphisms (SNPs) associated with CAD/MI. One of the most robust CAD/MI genetic associations is with intronic SNPs in the gene PHACTR1 on chromosome 6p24. How these PHACTR1 SNPs influence CAD/MI risk, and whether PHACTR1 itself is the causal gene at the locus, is currently unknown. APPROACH AND RESULTS: Using genetic fine-mapping and DNA resequencing experiments, we prioritized an intronic SNP (rs9349379) in PHACTR1 as causal variant. We showed that this variant is an expression quantitative trait locus for PHACTR1 expression in human coronary arteries. Experiments in endothelial cell extracts confirmed that alleles at rs9349379 are differentially bound by the transcription factors myocyte enhancer factor-2. We engineered a deletion of this myocyte enhancer factor-2-binding site using CRISPR/Cas9 genome-editing methodology. Heterozygous endothelial cells carrying this deletion express 35% less PHACTR1. Finally, we found no evidence that PHACTR1 expression levels are induced when stimulating human endothelial cells with vascular endothelial growth factor, tumor necrosis factor-α, or shear stress. CONCLUSIONS: Our results establish a link between intronic SNPs in PHACTR1, myocyte enhancer factor-2 binding, and transcriptional functions at the locus, PHACTR1 expression levels in coronary arteries and CAD/MI risk. Because PHACTR1 SNPs are not associated with the traditional risk factors for CAD/MI (eg, blood lipids or pressure, diabetes mellitus), our results suggest that PHACTR1 may influence CAD/MI risk through as yet unknown mechanisms in the vascular endothelium.