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Metabolic dysfunction, partly driven by altered liver function, predisposes to coronary artery disease (CAD), but the role of liver in vulnerable atherosclerotic plaque development remains unclear. Here we produced hepatocyte-like cells (HLCs) from 27 induced pluripotent stem cell (iPSC) lines derived from 15 study subjects with stable CAD (n = 5), acute CAD (n = 5) or healthy controls (n = 5). We performed a miRNA microarray screening throughout the differentiation, as well as compared iPSC-HLCs miRNA profiles of the patient groups to identify miRNAs involved in the development of CAD. MicroRNA profile changed during differentiation and started to resemble that of the primary human hepatocytes. In the microarray, 35 and 87 miRNAs were statistically significantly deregulated in the acute and stable CAD patients, respectively, compared to controls. Down-regulation of miR-149-5p, -92a-3p and -221-3p, and up-regulation of miR-122-5p was verified in the stable CAD patients when compared to other groups. The predicted targets of deregulated miRNAs were enriched in pathways connected to insulin signalling, inflammation and lipid metabolism. The iPSC-HLCs derived from stable CAD patients with extensive lesions had a distinct genetic miRNA profile possibly linked to metabolic dysfunction, potentially explaining the susceptibility to developing CAD. The iPSC-HLCs from acute CAD patients with only the acute rupture in otherwise healthy coronaries did not present a distinct miRNA profile, suggesting that hepatic miRNAs do not explain susceptibility to plaque rupture., (© 2023. The Author(s).)

Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) provide a renewable source of cells for drug discovery, disease modelling and cell-based therapies. Here, by using GRO-Seq we provide the first genome-wide analysis of the nascent RNAs in iPSCs, HLCs and primary hepatocytes to extend our understanding of the transcriptional changes occurring during hepatic differentiation process. We demonstrate that a large fraction of hepatocyte-specific genes are regulated at transcriptional level and identify hundreds of differentially expressed non-coding RNAs (ncRNAs), including primary miRNAs (pri-miRNAs) and long non-coding RNAs (lncRNAs). Differentiation induced alternative transcription start site (TSS) usage between the cell types as evidenced for miR-221/222 and miR-3613/15a/16-1 clusters. We demonstrate that lncRNAs and coding genes are tightly co-expressed and could thus be co-regulated. Finally, we identified sets of transcriptional regulators that might drive transcriptional changes during hepatocyte differentiation. These included RARG, E2F1, SP1 and FOXH1, which were associated with the down-regulated transcripts, and hepatocyte-specific TFs such as FOXA1, FOXA2, HNF1B, HNF4A and CEBPA, as well as RXR, PPAR, AP-1, JUNB, JUND and BATF, which were associated with up-regulated transcripts. In summary, this study clarifies the role of regulatory ncRNAs and TFs in differentiation of HLCs from iPSCs.

The apolipoprotein E (APOE) epsilon4 allele is associated with elevated cholesterol and risk of atherosclerosis. However, its role in ischemic stroke (IS) remains controversial. We investigated a possible link between IS or the severity of intracranial atherosclerosis and the APOE promoter polymorphisms -219G/T and +113G/C, involved in regulating APOE transcription. We genotyped subjects from a multicentric Belgian case-control study, including 237 middle-aged patients with IS due to small- or large-vessel atherosclerotic stroke and 326 ethnicity- and gender-matched controls and a Finnish autopsy series of 1004 non-stroke cases, who had received a quantitative score of atherosclerosis in the circle of Willis. The APOE epsilon4+ genotype did not associate with IS, but was related to more severe intracranial atherosclerosis score in men (5.4 vs 4.6, P=0.044). Within the most common APOE epsilon3/epsilon3 genotype group, the risk of IS associated with the G-allele of the tightly linked -219G/T (OR=6.2; 95% CI: 1.6-24.3, P=0.009) and +113G/C (OR=7.1; 95% CI: 1.7-29.9, P=0.007) promoter polymorphisms. There was no difference in the severity of intracranial atherosclerosis between -219G/G genotype carriers and non-carriers. This study suggests a multifaceted role of apoE on the risk of cerebrovascular diseases. The APOE epsilon4+ genotype did not predict the risk of IS but was associated with severity of subclinical intracranial atherosclerosis in men on the autopsy study. In contrast, the promoter variants were significant predictors of IS, suggesting that quantitative rather than qualitative variation of apoE is related to IS.