In vivo characterization of human APOA5 haplotypes.

Increased plasma triglyceride concentrations are an independent risk factor for cardiovascular disease. Numerous studies support a reproducible genetic association between two minor haplotypes in the human apolipoprotein A5 gene (APOA5) and increased plasma triglyceride concentrations. We thus sought to investigate the effects of these minor haplotypes (APOA5*2 and APOA5*3) on ApoAV plasma levels through the precise insertion of single-copy APOA5 haplotypes at a targeted location (Hprt) in the mouse genome. While we found no difference in the amount of human plasma ApoAV in mice containing the common APOA5*1 or minor APOA5*2 haplotype, the introduction of the single APOA5*3-defining allele (19W) resulted in three fold lower ApoAV plasma levels, consistent with existing genetic association studies. These results indicate that the S19W polymorphism is likely to be functional and explain the strong association of this variant with plasma triglycerides, supporting the value of sensitive in vivo assays to define the functional nature of human haplotypes.

Characterization of a new mouse model for human apolipoprotein A-I/C-III/A-IV deficiency.

Human data raised the possibility that coronary heart disease is associated with mutations in the apolipoprotein gene cluster APOA1/C3/A4 that result in multideficiency of cluster-encoded apolipoproteins and hypoalphalipoproteinemia. To test this hypothesis, we generated a mouse model for human apolipoprotein A-I (apoA-I)/C-III/A-IV deficiency. Homozygous mutants (Apoa1/c3/a4(-/-)) lacking the three cluster-encoded apolipoproteins were viable and fertile. In addition, feeding behavior and growth were apparently normal. Total cholesterol (TC), high density lipoprotein cholesterol (HDLc), and triglyceride levels in the plasma of fasted mutants fed a regular chow were 32% (P < 0.001), 17% (P < 0.001), and 70% (P < 0.01), respectively, those of wild-type mice. When fed a high-fat Western-type (HFW) diet, Apoa1/c3/a4(-/-) mice showed a further decrease in HDLc concentration and a moderate increase in TC, essentially in non-HDL fraction. The capacity of Apoa1/c3/a4(-/-) plasma to promote cholesterol efflux in vitro was decreased to 75% (P < 0.001), and LCAT activity was decreased by 38% (P < 0.01). Despite the very low total plasma cholesterol, the imbalance in lipoprotein distribution caused small but detectable aortic lesions in one-third of Apoa1/c3/a4(-/-) mice fed a HFW diet. In contrast, none of the wild-type mice had lesions. These results demonstrate that Apoa1/c3/a4(-/-) mice display clinical features similar to human apoA-I/C-III/A-IV deficiency (i.e., marked hypoalphalipoproteinemia) and provide further support for the apoa1/c3/a4 gene cluster as a minor susceptibility locus for atherosclerosis in mice.

Molecular mechanism of the blockade of plasma cholesteryl ester transfer protein by its physiological inhibitor apolipoprotein CI.

Genetically engineered mice demonstrated that apolipoprotein (apo) CI is a potent, physiological inhibitor of plasma cholesteryl ester transfer protein (CETP) activity. The goal of this study was to determine the molecular mechanism of the apoCI-mediated blockade of CETP activity. Kinetic analyses revealed that the inhibitory property of apoCI is independent of the amount of active CETP, but it is tightly dependent on the amount of high density lipoproteins (HDL) in the incubation mixtures. The electrostatic charge of HDL, i.e. the main carrier of apoCI in human plasma, is gradually modified with increasing amounts of apoCI, and the neutralization of apoCI lysine residues by acetylation produces a marked reduction in its inhibitory potential. The inhibitory property of full-length apoCI is shared by its C-terminal alpha-helix with significant electrostratic properties, whereas its N-terminal alpha-helix with no CETP inhibitory property has no effect on HDL electronegativity. Finally, binding experiments demonstrated that apoCI and to a lower extent its C-terminal alpha-helix are able to disrupt CETP-lipoprotein complexes in a concentration-dependent manner. It was concluded that the inhibition of CETP activity by apoCI is in direct link with its specific electrostatic properties, and the apoCI-mediated reduction in the binding properties of lipoproteins results in weaker CETP-HDL interactions and fewer cholesteryl ester transfers.

The ligand-binding function of hepatic lipase modulates the development of atherosclerosis in transgenic mice.

To investigate the separate contributions of the lipolytic versus ligand-binding function of hepatic lipase (HL) to plasma lipoprotein metabolism and atherosclerosis, we compared mice expressing catalytically active wild-type HL (HL-WT) and inactive HL (HL-S145G) with no endogenous expression of mouse apoE or HL (E-KO x HL-KO, where KO is knockout). HL-WT and HL-S145G reduced plasma cholesterol (by 40 and 57%, respectively), non-high density lipoprotein cholesterol (by 48 and 61%, respectively), and apoB (by 36 and 44%, respectively) (p < 0.01), but only HL-WT decreased high density lipoprotein cholesterol (by 67%) and apoA-I (by 54%). Compared with E-KO x HL-KO mice, both active and inactive HL lowered the pro-atherogenic lipoproteins by enhancing the catabolism of autologous (125)I-apoB very low density/intermediate density lipoprotein (VLDL/IDL) (fractional catabolic rates of 2.87 +/- 0.04/day for E-KO x HL-KO, 3.77 +/- 0.03/day for E-KO x HL-WT, and 3.63 +/- 0.09/day for E-KO x HL-S145G mice) and (125)I-apoB-48 low density lipoprotein (LDL) (fractional catabolic rates of 5.67 +/- 0.34/day for E-KO x HL-KO, 18.88 +/- 1.72/day for E-KO x HL-WT, and 9.01 +/- 0.14/day for E-KO x HL-S145G mice). In contrast, the catabolism of apoE-free, (131)I-apoB-100 LDL was not increased by either HL-WT or HL-S145G. Infusion of the receptor-associated protein (RAP), which blocks LDL receptor-related protein function, decreased plasma clearance and hepatic uptake of (131)I-apoB-48 LDL induced by HL-S145G. Despite their similar effects on lowering pro-atherogenic apoB-containing lipoproteins, HL-WT enhanced atherosclerosis by up to 50%, whereas HL-S145G markedly reduced aortic atherosclerosis by up to 96% (p < 0.02) in both male and female E-KO x HL-KO mice. These data identify a major receptor pathway (LDL receptor-related protein) by which the ligand-binding function of HL alters remnant lipoprotein uptake in vivo and delineate the separate contributions of the lipolytic versus ligand-binding function of HL to plasma lipoprotein size and metabolism, identifying an anti-atherogenic role of the ligand-binding function of HL in vivo.

Farnesoid X receptor agonists suppress hepatic apolipoprotein CIII expression.

BACKGROUND & AIMS: Increased serum triglyceride levels constitute a risk factor for coronary heart disease. Apolipoprotein CIII (Apo CIII) is a determinant of serum triglyceride metabolism. In this study, we investigated whether activators of the nuclear farnesoid X receptor (FXR) modulate Apo CIII gene expression. METHODS: The influence of bile acids and synthetic FXR activators on Apo CIII and triglyceride metabolism was studied in vivo by using FXR wild-type and FXR-deficient mice and in vitro by using human primary hepatocytes and HepG2 cells. RESULTS: In mice, treatment with the FXR agonist taurocholic acid strongly decreased serum triglyceride levels, an effect associated with reduced Apo CIII serum and liver messenger RNA levels. By contrast, no change was observed in FXR-deficient mice. Incubation of human primary hepatocytes and HepG2 cells with bile acids or the nonsteroidal synthetic FXR agonist GW4064 resulted in a dose-dependent down-regulation of Apo CIII gene expression. Promoter transfection experiments and mutation analysis showed that bile acid-activated FXR decrease human Apo CIII promoter activity via a negative FXR response element located in the I(4) footprint between nucleotides -739 and -704. Chromatin immunoprecipitation experiments showed that bile acid treatment led to binding of FXR/retinoid X receptor heterodimers to and displacement of HNF4alpha from this site. Bile acid treatment still repressed liver Apo CIII gene expression in hepatic HNF4alpha-deficient mice, suggesting an active rather than a competitive mechanism of Apo CIII repression by the FXR. CONCLUSIONS: We identified bile acid and synthetic activators of the nuclear FXR as negative regulators of Apo CIII expression, an effect that may contribute to the triglyceride-decreasing action of FXR agonists.

Transcriptional regulation of human Rev-erbalpha gene expression by the orphan nuclear receptor retinoic acid-related orphan receptor alpha.

The Rev-erb and retinoic acid-related orphan receptors (ROR) are two related families of orphan nuclear receptors that recognize similar response elements but have opposite effects on transcription. Recently, the Rev-erbalpha gene promoter has been characterized and shown to harbor a functional Rev-erbalpha-binding site known as Rev-DR2, responsible for negative feedback down-regulation of promoter activity by Rev-erbalpha itself. The present study aimed to investigate whether Rev-erbalpha gene expression is regulated by RORalpha. Gel shift analysis demonstrated that in vitro translated hRORalpha1 protein binds to the Rev-DR2 site, both as monomer and dimer. Chromatin immunoprecipitation assays demonstrated that binding of RORalpha to this site also occurred in vivo in human hepatoma HepG2 cells. The Rev-DR2 site was further shown to be functional as it conferred hRORalpha1 responsiveness to a heterologous promoter and to the natural human Rev-erbalpha gene promoter in these cells. Mutation of this site in the context of the natural Rev-erbalpha gene promoter abolished its activation by RORalpha, indicating that this site plays a key role in hRORalpha1 action. Finally, adenoviral overexpression of hRORalpha1 in HepG2 cells led to enhanced hRev-erbalpha mRNA accumulation, further confirming the physiological importance of RORalpha1 in the regulation of Rev-erbalpha expression.

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL/6 and apoE-knockout mice.

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in C57BL/6 mice resulted in a unique anti-atherogenic profile characterized by decreased plasma cholesterol (63%), cholesteryl ester (63%), free cholesterol (67%), non-high density lipoprotein (HDL)-cholesterol (53%), and apolipoprotein (apo) B (64%) but markedly increased HDL-cholesterol (2.8-fold), apoA-I (2.2-fold), and apoE (2.8-fold) levels. These beneficial changes in the lipid profile led to significantly lower (65%) aortic atherosclerosis in hABCA1-Tg mice. In marked contrast, ABCA1 overexpression had a minimal effect on the plasma lipid profile of apoE-KO mice and resulted in a 2- to 2.6-fold increase in aortic lesion area. These combined results indicate that overexpression of ABCA1 in C57BL/6 mice on a high cholesterol diet results in an atheroprotective lipoprotein profile and decreased atherosclerosis, and thus provide previously undocumented in vivo evidence of an anti-atherogenic role for the ABCA1 transporter. In contrast, overexpression of ABCA1 in an apoE-KO background led to increased atherosclerosis, further substantiating the important role of apoE in macrophage cholesterol metabolism and atherogenesis. In summary, these results establish that, in the presence of apoE, overexpression of ABCA1 modulates HDL as well as apoB-containing lipoprotein metabolism and reduces atherosclerosis in vivo, and indicate that pharmacological agents that will increase ABCA1 expression may reduce atherogenic risk in humans.