Allele-specific expression (ASE) analysis, which quantifies the relative expression of two alleles in a diploid individual, is a powerful tool for identifying cis-regulated gene expression variations that underlie phenotypic differences among individuals. Existing methods for gene-level ASE detection analyze one individual at a time, therefore failing to account for shared information across individuals. Failure to accommodate such shared information not only reduces power, but also makes it difficult to interpret results across individuals. However, when only RNA sequencing (RNA-seq) data are available, ASE detection across individuals is challenging because the data often include individuals that are either heterozygous or homozygous for the unobserved cis-regulatory SNP, leading to sample heterogeneity as only those heterozygous individuals are informative for ASE, whereas those homozygous individuals have balanced expression. To simultaneously model multi-individual information and account for such heterogeneity, we developed ASEP, a mixture model with subject-specific random effect to account for multi-SNP correlations within the same gene. ASEP only requires RNA-seq data, and is able to detect gene-level ASE under one condition and differential ASE between two conditions (e.g., pre- versus post-treatment). Extensive simulations demonstrated the convincing performance of ASEP under a wide range of scenarios. We applied ASEP to a human kidney RNA-seq dataset, identified ASE genes and validated our results with two published eQTL studies. We further applied ASEP to a human macrophage RNA-seq dataset, identified genes showing evidence of differential ASE between M0 and M1 macrophages, and confirmed our findings by results from cardiometabolic trait-relevant genome-wide association studies. To the best of our knowledge, ASEP is the first method for gene-level ASE detection at the population level that only requires the use of RNA-seq data. With the growing adoption of RNA-seq, we believe ASEP will be well-suited for various ASE studies for human diseases.
<p dir="ltr">Insulin activates insulin receptor (IR) signaling and subsequently triggers IR endocytosis to attenuate signaling. Cell division regulators MAD2, BUBR1, and p31<sup>comet</sup> promote IR endocytosis upon insulin stimulation. Here, we show that genetic ablation of the IR-MAD2 interaction in mice delays IR endocytosis, increases IR levels, and prolongs insulin action at the cell surface. This in turn causes a defect in insulin clearance and increases circulating insulin levels, unexpectedly increasing glucagon levels, which alters glucose metabolism modestly. Disruption of the IR–MAD2 interaction increases serum fatty acid concentrations and hepatic fat accumulation in fasted male mice. Furthermore, disruption of the IR–MAD2 interaction distinctly changes metabolic and transcriptomic profiles in the liver and adipose tissues. Our findings establish the function of cell division regulators in insulin signaling and provide insights into the metabolic functions of IR endocytosis.</p><p><br></p><p dir="ltr"><b>Article highlights</b></p><p dir="ltr">· Physiological function of IR trafficking on insulin sensitivity remains unresolved.</p><p dir="ltr">· Disruption of the IR-MAD2 interaction delays IR endocytosis and prolongs insulin signaling.</p><p dir="ltr">· IR-MAD2 controls insulin clearance and glucose metabolism.</p><p dir="ltr">· IR-MAD2 maintains energy homeostasis.</p>
Tangier disease (TD) is an autosomal recessive disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1). These result in a greatly reduced ability to transport cholesterol out of cells, leading to the accumulation of cholesterol in macrophages and many other body tissues expressing ABCA1. We recruited two TD individuals, TD1, compound heterozygote at S2046R/K531N, and TD2, homozygous for the E1005X/E1005X truncation mutation, as well as heterozygote/non-affected family members, and healthy controls. We reprogrammed PBMC of TD patients and controls to pluripotency by Sendai viral transduction with Oct3/4, Sox2, Klf4 and c-Myc. TD-iPSC expressed pluripotency markers including SSEA-3, SSEA-4, TRA-1.60, and TRA-1.81, and maintained a normal karyotype. TD iPSC differentiated efficiently to macrophages. iPSC-derived macrophages (IPSDM) characteristics paralleled those of primary PBMC-derived macrophages (HMDM) at morphological, phenotypic and transcriptomic levels. TD-IPSDM and HMDM showed no cholesterol efflux to apolipoprotein A-I (apoA-I) and impaired efflux to HDL 3 . Treatment of TD cells with LXR agonists, which upregulate ABCA1 expression, failed to enhance cholesterol efflux to apoA-I in TD-IPSDM and HMDM consistent with the absence of functional ABCA1. In both IPSDM and HMDM, the heterozygote ABCA1 mutation carrier had an intermediate defect in cholesterol efflux and a partial response to the LXR agonist consistent with the presence of one functional allele. Relative to control-IPSDM, TD-IPSDM also showed a higher cholesterol ester/total cholesterol (CE/TC) ratio upon acetylated-LDL loading. Compared with control-IPSDM, TD-IPSDM showed enhanced phagocytosis of zymosan particles and greater inflammatory response to ATP treatment in lipopolysaccharide (LPS)-primed IPSDM, evidenced by markedly elevated gene expression of IL-1beta, IL-6, IL-8 and CCL5, but not TNF-alpha. These observations provide further support for the utility of IPSDM in defining more subtle macrophage phenotypes that are less obvious manifestations of Mendelian disorders. We conclude that macrophages derived from TD IPS can effectively recapitulate pathologic hallmarks of the disease.
Macrophages fulfill homeostatic functions beyond defense. Monocytes/macrophages derived from human induced pluripotent stem cells (hiPSCs) are useful tools to study vascular diseases and potential sources of cell-based therapies. The objective of this study is to generate PBMC-hiPSC derived monocytes, macrophages, as well as M1 and M2 type differentiated macrophages and to examine multiple macrophage characteristics of relevance to cardio-metabolic disease. We briefly cultured human adult PBMCs from healthy volunteers to enrich for the erythroid progenitors. These progenitors were used to generate hiPSCs using lentivirus-mediated transduction of Oct3/4, Sox2, Klf4 and c-Myc. A stepwise protocol was used for the differentiation of PBMC-hiPSCs to monocytes/macrophages. The protocol involves primitive streak and mesoderm induction (stage 1), hematopoietic specification (stage 2), hematopoietic cell maturation and myeloid expansion (stage 3), monocytes collection and differentiation to macrophages (stage 4). Differentiation cultures were set up in six-well plate format, from 20 embryoid bodies per well, and produced up to 30 million macrophages. hiPSC-derived macrophages (hiPSC-MΦ) exhibited spindle morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of hiPSC-MΦ revealed positive staining for CD68, CD11b, and CCL2. FACS analysis indicates >90% expression of macrophage lineage markers, including CD45, CD14, CD16, CD115, CD68, CX3CR1, CD11b, and CD18. Migration of hiPSC-MΦ in response to M-CSF was confirmed by Boyden Chamber assay. hiPSC-MΦ were polarized to M1 (classical activation stimulated by TLR ligands LPS and IFN-gamma) and M2 (alternative activation stimulated by IL-4) types. M2 macrophages were characterized by more efficient phagocytic activity of zymosan particles and greater migration capacities compared with M1 macrophages. The results demonstrated the feasibility of differentiation/polarization of macrophages from PBMC-hiPSCs. This study advanced our understanding of the suitability of subject-specific hiPSC for deriving myeloid/monocyte/macrophages for study of monocyte-macrophage functions of direct relevance to cardio-metabolic disease.
We adopted a transcriptome-wide microarray analysis approach to determine the extent to which vascular gene expression is altered as a result of juvenile obesity and identify obesity-responsive mRNAs. We examined transcriptional profiles in the left anterior descending coronary artery (LAD), perivascular fat adjacent to the LAD, and descending thoracic aorta between obese (n = 5) and lean (n = 6) juvenile Ossabaw pigs (age = 22 wk). Obesity was experimentally induced by feeding the animals a high-fat/high-fructose corn syrup/high-cholesterol diet for 16 wk. We found that expression of 189 vascular cell genes in the LAD and expression of 165 genes in the thoracic aorta were altered with juvenile obesity (false discovery rate ≤ 10%) with an overlap of only 28 genes between both arteries. Notably, a number of genes found to be markedly upregulated in the LAD of obese pigs are implicated in atherosclerosis, including ACP5, LYZ, CXCL14, APOE, PLA2G7, LGALS3, SPP1, ITGB2, CYBB, and P2RY12. Furthermore, pathway analysis revealed the induction of proinflammatory and pro-oxidant pathways with obesity primarily in the LAD. Gene expression in the LAD perivascular fat was minimally altered with juvenile obesity. Together, we provide new evidence that obesity produces artery-specific changes in pretranslational regulation with a clear upregulation of proatherogenic genes in the LAD. Our data may offer potential viable drug targets and mechanistic insights regarding the molecular precursors involved in the origins of overnutrition and obesity-associated vascular disease. In particular, our results suggest that the oxidized LDL/LOX-1/NF-κB signaling axis may be involved in the early initiation of a juvenile obesity-induced proatherogenic coronary artery phenotype.
C irculating lipoproteins containing cholesterol and tri- glycerides are major risk factors for many cardiovascular diseases (CVD).Many therapeutic strategies targeting lipid metabolism have been successfully used to treat dyslipidemia and subsequently reduce cardiovascular risk.Most notably, statins, which effectively lower low-density lipoprotein-cholesterol (LDL-C), have been widely used and have been shown to substantially reduce cardiovascular events.More recently, a new class of LDL-lowering drugs, the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, has been developed with highly promising results.Many other therapeutic approaches aiming to modulate lipids are also being considered.These include antisense oligonucleotides (ASOs) designed to inhibit well-characterized gene targets involved in dyslipidemia, modulators of high-density lipoprotein (HDL) function and reverse cholesterol transport (RCT) pathways, and triglyceride-lowering drugs.We herein highlight recent basic, translational, and clinical studies published in ATVB that further elucidates the mechanisms and therapeutic potential of these approaches. PCSK9: A New Promising Therapeutic TargetLDL-receptor (LDLR) is a cell surface receptor that was found to be mutated in patients with familial hypercholesterolemia (FH). 1 FH is a genetic disorder typically characterized by hypercholesterolemia, ectopic cholesterol deposition, and premature CVD.Hepatic LDLR is essential for the uptake of circulating LDL-C, which cannot be effectively cleared in FH patients.Conversely, increasing LDLR levels, particularly in the liver, enhances LDL-C clearance.Recent evidence identified PCSK9, a protein secreted by the liver, as a mediator of LDLR degradation by interacting with its extracellular domain and promoting its trafficking to the lysosome, rather than allowing normal recycling to the cell surface. 2,3PCSK9 gain-of-function mutations in humans were associated with decreased LDLR levels, and thus increased circulating LDL-C, causing autosomal dominant hypercholesterolemia. 4,5 In contrast, loss-of-function mutations in PCSK9 were associated with lower LDL-C levels and reduced CVD events. 6Therefore, PCSK9 became a promising therapeutic target. 7his section highlights the endogenous and exogenous modulators of PCSK9 levels, its pleiotropic effects, and the clinical implications of PCSK9 mutations and circulating levels.
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