Smooth muscle cells in human atherosclerosis: proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease.

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hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver

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Methylation status of nc886 epiallele reflects periconceptional conditions and is associated with glucose metabolism through nc886 RNAs

BackgroundNon-coding RNA 886 (nc886) is coded from a maternally inherited metastable epiallele. We set out to investigate the determinants and dynamics of the methylation pattern at the nc886 epiallele and how this methylation status associates with nc886 RNA expression. Furthermore, we investigated the associations between the nc886 methylation status or the levels of nc886 RNAs and metabolic traits in the YFS and KORA cohorts. The association between nc886 epiallele methylation and RNA expression was also validated in induced pluripotent stem cell (iPSC) lines.ResultsWe confirm that the methylation status of the nc886 epiallele is mostly binomial, with individuals displaying either a non- or hemi-methylated status, but we also describe intermediately and close to fully methylated individuals. We show that an individual's methylation status is associated with the mother's age and socioeconomic status, but not with the individual's own genetics. Once established, the methylation status of the nc886 epiallele remains stable for at least 25 years. This methylation status is strongly associated with the levels of nc886 non-coding RNAs in serum, blood, and iPSC lines. In addition, nc886 methylation status associates with glucose and insulin levels during adolescence but not with the indicators of glucose metabolism or the incidence of type 2 diabetes in adulthood. However, the nc886-3p RNA levels also associate with glucose metabolism in adulthood.ConclusionsThese results indicate that nc886 metastable epiallele methylation is tuned by the periconceptional conditions and it associates with glucose metabolism through the expression of the ncRNAs coded in the epiallele region.</p

Coronary artery disease patient-derived iPSC-hepatocytes have distinct miRNA profile that may alter lipid metabolism

Abstract 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

Lipidomic profiling of patient-specific iPSC-derived hepatocyte-like cells

Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (iPSCs) offer an alternative model to primary human hepatocytes to study lipid aberrations. However, the detailed lipid profile of HLCs is yet unknown. In the current study, functional HLCs were differentiated from iPSCs generated from dermal fibroblasts of three individuals by a three-step protocol through the definitive endoderm (DE) stage. In parallel, detailed lipidomic analyses as well as gene expression profiling of a set of lipid-metabolism-related genes were performed during the entire differentiation process from iPSCs to HLCs. Additionally, fatty acid (FA) composition of the cell culture media at different stages was determined. Our results show that major alterations in the molecular species of lipids occurring during DE and early hepatic differentiation stages mainly mirror the quality and quantity of the FAs supplied in culture medium at each stage. Polyunsaturated phospholipids and sphingolipids with a very long FA were produced in the cells at a later stage of differentiation. This work uncovers the previously unknown lipid composition of iPSC-HLCs and its alterations during the differentiation in conjunction with the expression of key lipid-associated genes. Together with biochemical, functional and gene expression measurements, the lipidomic analyses allowed us to improve our understanding of the concerted influence of the exogenous metabolite supply and cellular biosynthesis essential for iPSC-HLC differentiation and function. Importantly, the study describes in detail a cell model that can be applied in exploring, for example, the lipid metabolism involved in the development of fatty liver disease or atherosclerosis.Peer reviewe

Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the halflife, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than longchain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.publishedVersionPeer reviewe

Improvements in Maturity and Stability of 3D iPSC-Derived Hepatocyte-like Cell Cultures

Induced pluripotent stem cell (iPSC) technology enables differentiation of human hepatocytes or hepatocyte-like cells (iPSC-HLCs). Advances in 3D culturing platforms enable the development of more in vivo-like liver models that recapitulate the complex liver architecture and functionality better than traditional 2D monocultures. Moreover, within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation and maintenance of hepatocyte metabolic function. Thus, models combining 3D culture and co-culturing of various cell types potentially create more functional in vitro liver models than 2D monocultures. Here, we report the establishment of 3D cultures of iPSC-HLCs alone and in co-culture with human umbilical vein endothelial cells (HUVECs) and adipose tissue-derived mesenchymal stem/stromal cells (hASCs). The 3D cultures were performed as spheroids or on microfluidic chips utilizing various biomaterials. Our results show that both 3D spheroid and on-chip culture enhance the expression of mature liver marker genes and proteins compared to 2D. Among the spheroid models, we saw the best functionality in iPSC-HLC monoculture spheroids. On the contrary, in the chip system, the multilineage model outperformed the monoculture chip model. Additionally, the optical projection tomography (OPT) and electrical impedance tomography (EIT) system revealed changes in spheroid size and electrical conductivity during spheroid culture, suggesting changes in cell–cell connections. Altogether, the present study demonstrates that iPSC-HLCs can successfully be cultured in 3D as spheroids and on microfluidic chips, and co-culturing iPSC-HLCs with NPCs enhances their functionality. These 3D in vitro liver systems are promising human-derived platforms usable in various liver-related studies, specifically when using patient-specific iPSCs.Peer reviewe

Associations of apolipoprotein E gene with ischemic stroke and intracranial atherosclerosis.

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.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe