Large, Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci.

Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.

Identification of the active form of endothelial lipase, a homodimer in a head-to-tail conformation.

Endothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation.

Complete prevention of atherosclerosis in apoE-deficient mice by hepatic human apoE gene transfer with adeno-associated virus serotypes 7 and 8.

OBJECTIVE: Using intravenous injection of adeno-associated viral (AAV) vectors based on novel serotypes 7 and 8, we examined whether liver-specific expression of human apolipoprotein E (apoE) in apoE-deficient mice would completely prevent atherosclerosis after 1 year of sustained expression. METHODS AND RESULTS: Chow-fed apoE-/- mice were injected via the tail vein with vectors based on AAV2 or novel serotypes AAV7 and AAV8 encoding human apoE3 driven by a liver-specific promoter. In contrast to the first-generation AAV2 vector, apoE levels of mice injected with chimeric AAV2/7 and AAV2/8 vectors reached approximately 2-fold greater than normal human plasma levels by week 4 and maintained therapeutic levels up to 1 year. Cholesterol levels of AAV2/7-apoE and AAV2/8-apoE-treated mice were reduced to normal murine wild-type levels and were maintained for 1 year. At termination after 1 year, extensive atherosclerosis was present in the thoracic aortas and aortic roots of control AAV2/8-lacZ and AAV2-apoE-injected mice, but was completely prevented in both the AAV2/7 and AAV2/8-apoE-treated mice. CONCLUSIONS: We demonstrate that intravenous administration of AAV2/7- and AAV2/8-apoE vectors effectively mediated robust and sustained hepatic-specific expression of apoE and completely prevented atherosclerosis at 1 year.

Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras.

The triglyceride (TG) lipase gene subfamily, consisting of LPL, HL, and endothelial lipase (EL), plays a central role in plasma lipoprotein metabolism. Compared with LPL and HL, EL is relatively more active as a phospholipase than as a TG lipase. The amino acid loop or "lid" covering the catalytic site has been implicated as the basis for the difference in substrate specificity between HL and LPL. To determine the role of the lid in the substrate specificity of EL, we studied EL in comparison with LPL by mutating specific residues of the EL lid and exchanging their lids. Mutation studies showed that amphipathic properties of the lid contribute to substrate specificity. Exchanging lids between LPL and EL only partially shifted the substrate specificity of the enzymes. Studies of a double chimera possessing both the lid and the C-terminal domain (C-domain) of EL in the LPL backbone showed that the role of the lid in determining substrate specificity does not depend on the nature of the C-domain of the lipase. Using a kinetic assay, we showed an additive effect of the EL lid on the apparent affinity for HDL(3) in the presence of the EL C-domain.

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1.

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway.

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.