A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitro differentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.
A new member of the lipase gene family, initially termed endothelial lipase (gene nomenclature, LIPG; protein, EL), is expressed in a variety of different tissues, suggesting a general role in lipid metabolism. To assess the hypothesis that EL plays a physiological role in lipoprotein metabolism in vivo, we have used gene targeting of the native murine locus and transgenic introduction of the human LIPG locus in mice to modulate the level of EL expression. Evaluation of these alleles in a C57Bl/6 background revealed an inverse relationship between HDL cholesterol level and EL expression. Fasting plasma HDL cholesterol was increased by 57% in LIPG–/– mice and 25% in LIPG+/– mice and was decreased by 19% in LIPG transgenic mice as compared with syngeneic controls. Detailed analysis of lipoprotein particle composition indicated that this increase was due primarily to an increased number of HDL particles. Phospholipase assays indicated that EL is a primary contributor to phospholipase activity in mouse. These data indicate that expression levels of this novel lipase have a significant effect on lipoprotein metabolism
Endothelial lipase (EL) expression correlates inversely with circulating high density lipoprotein (HDL) cholesterol levels in genetic mouse models, and human genetic variation in this locus has been linked to differences in HDL cholesterol levels. These data suggest a role for EL in the development of atherosclerotic vascular disease. To investigate this possibility, LIPG-null alleles were bred onto the apoE knockout background, and the homozygous double knockout animals were characterized. Both apoE knockout and double knockout mice had low HDL cholesterol levels when compared with wild-type mice, but the HDL cholesterol levels of the double knockout mice were higher than those of apoE knockout mice. Atherogenic very low density lipoprotein and intermediate density lipoprotein/low density lipoprotein cholesterol levels of the double knockout mice were also greater than those of the apoE knockout animals. Despite this lipid profile, there was a significant ϳ70% decrease in atherosclerotic disease area in double knockout mice on a regular diet. Immunohistochemistry and protein blot studies revealed increased EL expression in the atherosclerotic aortas of the apoE knockout animals. An observed decrease in macrophage content in vessels lacking EL correlated with ex vivo vascular monocyte adhesion assays, suggesting that this protein can modulate monocyte adhesion and infiltration into diseased tissues. These data suggest that EL may have indirect atherogenic actions in vivo through its effect on circulating HDL cholesterol and direct atherogenic actions through vascular wall processes such as monocyte recruitment and cholesterol uptake.
Objective-ApoAV, a newly discovered apoprotein, affects plasma triglyceride level. To determine how this occurs, we studied triglyceride-rich lipoprotein (TRL) metabolism in mice deficient in apoAV. Methods and Results-No significant difference in triglyceride production rate was found between apoa5 Ϫ/Ϫ mice and controls. The presence or absence of apoAV affected TRL catabolism. After the injection of 14 C-palmitate and 3 H-cholesterol labeled chylomicrons and 125 I-labeled chylomicron remnants, the disappearance of 14 C, 3 H, and 125 I was significantly slower in apoa5 Ϫ/Ϫ mice relative to controls. This was because of diminished lipolysis of TRL and the reduced rate of uptake of their remnants in apoa5 Ϫ/Ϫ mice. Observed elevated cholesterol level was caused by increased high-density lipoprotein (HDL) cholesterol in apoa5 Ϫ/Ϫ mice. VLDL from apoa5 Ϫ/Ϫ mice were poor substrate for lipoprotein lipase, and did not bind to the low-density lipoprotein (LDL) receptor as well as normal very-low-density lipoprotein (VLDL). LDL receptor levels were slightly elevated in apoa5 Ϫ/Ϫ mice consistent with lower remnant uptake rates. These alterations may be the result of the lower apoE-to-apoC ratio found in VLDL isolated from apoa5 Ϫ/Ϫ mice. Key Words: Apoa5 Ⅲ hypertriglyceridemia Ⅲ knockout Ⅲ lipolysis Ⅲ triglyceride-rich lipoproteins T he level of triglycerides in the blood has been correlated with the risk of atherosclerosis in a variety of studies. 1,2 Apoproteins of triglyceride-rich lipoproteins (TRL) play an important role in triglyceride transport. In particular, ApoCs and apoE 3,4 strongly affect TRL metabolism and thus plasma triglyceride levels. ApoAV is a newly discovered apolipoprotein, which was identified independently by 2 groups. 5,6 Disruption of the apoa5 gene in mice resulted in hypertriglyceridemia, whereas overexpression led to decreased plasma triglyceride concentrations, thus establishing an important role for this protein in triglyceride homeostasis. 5 One explanation for apoAV's lack of earlier identification is its low plasma concentration (Ϸ1 g/mL), which may be in part caused by the observation that during its synthesis, it is largely retained in the endoplasmic reticulum and does not traffic to the Golgi. 7,8 Furthermore, the low concentration of apoAV in plasma suggests that either this protein does not exert its effect in plasma or that it has a very potent effect on lipoprotein particle composition and/or metabolism. Several studies in humans 9 revealed strong associations between APOA5 polymorphisms and triglyceride levels. Most striking was the association between the minor APOA5 haplotypes found in 25% to 50% of whites, blacks, and Hispanics, and increased plasma triglyceride levels. To better understand the specific mechanisms by which altered apoAV exerts its effect on triglyceride levels in humans and mice, we studied the metabolism of TRL in vivo in mice lacking apoAV. Conclusions-These See page 2445ApoAV could influence triglyceride levels through alterations in the hepatic triglycer...
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