Searching for avidity by chemical ligation of combinatorially self-assembled DNA-encoded ligand libraries.

DNA encoded ligands are self-assembled into bivalent complexes and chemically ligated to link their identities. To demonstrate their potential as a combinatorial screening platform for avidity interactions, the optimal bivalent aptamer design (examplar ligands) for human alpha-thrombin is determined in a single round of selection and the DNA scaffold replaced with minimal impact on the final design.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Znf202 affects high density lipoprotein cholesterol levels and promotes hepatosteatosis in hyperlipidemic mice.

BACKGROUND: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. METHODOLOGY AND PRINCIPAL FINDINGS: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression. CONCLUSION/SIGNIFICANCE: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

Human ATP-binding cassette G1 controls macrophage lipoprotein lipase bioavailability and promotes foam cell formation.

OBJECTIVE: The physiological function of the ATP-binding cassette G1 (ABCG1) transporter in humans is not yet elucidated, as no genetic disease caused by ABCG1 mutations has been documented. The goal of our study was, therefore, to investigate the potential role(s) of ABCG1 in lipid metabolism in humans. METHODS AND RESULTS: Here we report that among the 104 polymorphisms present in the ABCG1 gene, the analysis of the frequent functional rs1893590 and rs1378577 single nucleotide polymorphisms located in the regulatory region of ABCG1 in the Regression Growth Evaluation Statin Study population revealed that both ABCG1 single nucleotide polymorphisms were significantly associated with plasma lipoprotein lipase (LPL) activity. Moreover, we observed that plasma LPL activity was modestly reduced in Abcg1(-/-) mice as compared with control mice. Adipose tissue and skeletal muscle are the major tissues accounting for levels and activity of plasma LPL in the body. However, beyond its lipolytic action in the plasma compartment, LPL was also described to act locally at the cellular level. Thus, macrophage LPL was reported to promote foam cell formation and atherosclerosis in vivo. Analysis of the relationship between ABCG1 and LPL in macrophages revealed that the knockdown of ABCG1 expression (ABCG1 knockdown) in primary cultures of human monocyte-derived macrophages using small interfering RNAs led to a marked reduction of both the secretion and activity of LPL. Indeed, LPL was trapped at the cell surface of ABCG1 knockdown human monocyte-derived macrophages, likely in cholesterol-rich domains, thereby reducing the bioavailability and activity of LPL. As a consequence, LPL-mediated lipid accumulation in human macrophage foam cells in the presence of triglyceride-rich lipoproteins was abolished when ABCG1 expression was repressed. CONCLUSIONS: We presently report that ABCG1 controls LPL activity and promotes lipid accumulation in human macrophages in the presence of triglyceride-rich lipoproteins, thereby suggesting a potential deleterious role of macrophage ABCG1 in metabolic situations associated with high levels of circulating triglyceride-rich lipoproteins together with the presence of macrophages in the arterial wall.

Effects of deletion of macrophage ABCA7 on lipid metabolism and the development of atherosclerosis in the presence and absence of ABCA1.

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen.

The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout mice depends on the stage of atherogenesis.

OBJECTIVE: As ABCG1 plays a role in cholesterol efflux, macrophage ABCG1 expression has been suggested to protect against atherosclerosis. However, we and others observed varying effects of ABCG1 deficiency on atherosclerotic lesion size. The objective of this study was to define the effect of ABCG1 deficiency during atherosclerotic lesion progression in LDL receptor knockout (LDLr(-/-)) mice. METHODS AND RESULTS: ABCG1(-/-)/LDLr(-/-) and ABCG1(+/+)/LDLr(-/-) littermates were fed a Western-type diet for 10 and 12 weeks in order to study the effect of ABCG1 deficiency in the exponential phase of atherosclerotic lesion formation. At 10 weeks of diet feeding, a significant 1.5-fold increase in early atherosclerotic lesion size (130±12×10(3) µm(2)) was observed in ABCG1(-/-)/LDLr(-/-) mice compared to ABCG1(+/+)/LDLr(-/-) mice (88±11×10(3) µm(2); p<0.05). Interestingly, in more advanced lesions, induced by 12 weeks of WTD feeding, ABCG1(-/-)/LDLr(-/-) mice showed a significant 1.7-fold decrease in atherosclerotic lesion size (160±20×10(3) µm(2) vs 273±19×10(3) µm(2) in control mice; p<0.01), indicating that in the ABCG1(-/-)/LDLr(-/-) mice progression of lesion formation is retarded as compared to ABCG1(+/+)/LDLr(-/-) mice. In addition, correlation analysis performed on 7 independent published studies and the current study confirmed that ABCG1 is atheroprotective in early lesions, while the development of advanced lesions is stimulated. CONCLUSIONS: It appears that the effect of ABCG1 deficiency on lesion development in LDLr(-/-) mice depends on the stage of atherogenesis, whereby the absence of ABCG1 leads to increased lesions at sizes<167×10(3) µm(2) while in more advanced stages of atherosclerosis enhanced apoptosis and/or compensatory mechanisms lead to retarded lesion progression.

Apolipoprotein isoform E4 does not increase coronary heart disease risk in carriers of low-density lipoprotein receptor mutations.

BACKGROUND: In humans, the E4 allele of the apolipoprotein E gene is associated with increased coronary heart disease risk. Surprisingly, in rodents, apolipoprotein E4 only accelerates the atherosclerotic process when transgenic for the human low-density lipoprotein receptor (LDLR) protein. We therefore investigated whether the LDLR locus interacted with the apolipoprotein E gene genotype on coronary heart disease risk in patients clinically diagnosed with familial hypercholesterolemia with and without LDLR mutation. We investigated whether the presence of an LDLR mutation diminishing LDLR function was protective in E4/E4 carriers. METHODS AND RESULTS: In a cohort of 2400 patients clinically diagnosed with familial hypercholesterolemia, we found an LDLR gene mutation in 1383 patients, whereas in 1013 patients, such mutation was not present. In 92 patients homozygous for the apolipoprotein E4, the presence of an LDLR mutation conferred lower coronary heart disease risk (hazard ratio, 0.16; 95% CI, 0.05-0.58; P=0.005). Mirroring these results, the apolipoprotein E4/E4 genotype was also associated with lower coronary heart disease risk in patients with familial hypercholesterolemia with an LDLR mutation (hazard ratio, 0.26; hazard ratio, 0.08-0.80; P=0.02). CONCLUSIONS: LDLR function is key to the detrimental effects of apolipoprotein E4 in humans. Kinetic studies in humans are now required to study the consequences of our observation for prevention of both coronary heart disease and Alzheimer disease.

Liver X receptors inhibit macrophage proliferation through downregulation of cyclins D1 and B1 and cyclin-dependent kinases 2 and 4.

Macrophages serve essential functions as regulators of immunity and homeostasis, and their proliferation contributes to pathogenesis of certain disorders. In this report, we show that induction of macrophage proliferation by the growth factor M-CSF is negatively modulated by agonists that activate the nuclear receptor liver X receptor (LXR), both in vitro and in vivo. Both isoforms LXR α and ß are involved in the antiproliferative actions of LXR ligands in macrophages. In contrast, M-CSF does not exert negative effects on LXR-mediated gene expression. Treatment with LXR agonists results in the accumulation of macrophages in the G(0)/G(1) phase of the cell cycle without affecting ERK-1/2 phosphorylation. The use of small interfering RNA or genetically modified mice revealed that, in contrast to other cellular models, functional expression of either the cyclin-dependent kinase inhibitor p27KIP1 or the cholesterol transporters ATP-binding cassette A1 or ATP-binding cassette G1 was not required for the antiproliferative effects of LXR agonists in macrophages. Western blot analysis revealed that protein expression of key molecules that regulate progression through the cell cycle, such as cyclins D1 and B1 and cyclin-dependent kinases 2 and 4, was downregulated upon LXR activation. These observations suggest a role for LXR agonists in limiting macrophage proliferative responses associated to pathogenic disorders.

Genetic variant of the scavenger receptor BI in humans.

BACKGROUND: In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It is unclear what role SR-BI plays in human metabolism. METHODS: We sequenced the gene encoding SR-BI in persons with elevated HDL cholesterol levels and identified a family with a new missense mutation (P297S). The functional effects of the P297S mutation on HDL binding, cellular cholesterol uptake and efflux, atherosclerosis, platelet function, and adrenal function were studied. RESULTS: Cholesterol uptake from HDL by primary murine hepatocytes that expressed mutant SR-BI was reduced to half of that of hepatocytes expressing wild-type SR-BI. Carriers of the P297S mutation had increased HDL cholesterol levels (70.4 mg per deciliter [1.8 mmol per liter], vs. 53.4 mg per deciliter [1.4 mmol per liter] in noncarriers; P<0.001) and a reduced capacity for efflux of cholesterol from macrophages, but the carotid artery intima-media thickness was similar in carriers and in family noncarriers. Platelets from carriers had increased unesterified cholesterol content and impaired function. In carriers, adrenal steroidogenesis was attenuated, as evidenced by decreased urinary excretion of sterol metabolites, a decreased response to corticotropin stimulation, and symptoms of diminished adrenal function. CONCLUSIONS: We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).

Macrophage adipose triglyceride lipase deficiency attenuates atherosclerotic lesion development in low-density lipoprotein receptor knockout mice.

OBJECTIVE: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis. METHODS AND RESULTS: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population. CONCLUSIONS: We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.

Enhanced foam cell formation, atherosclerotic lesion development, and inflammation by combined deletion of ABCA1 and SR-BI in Bone marrow-derived cells in LDL receptor knockout mice on western-type diet.

RATIONALE: macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-poor apolipoprotein AI, and scavenger receptor class B type I (SR-BI), which promotes efflux to mature high-density lipoprotein. OBJECTIVE: the aim of the present study was to increase the insight into the putative synergistic roles of ABCA1 and SR-BI in foam cell formation and atherosclerosis. METHODS AND RESULTS: low-density lipoprotein receptor knockout (LDLr KO) mice were transplanted with bone marrow from ABCA1/SR-BI double knockout mice, the respective single knockouts, or wild-type littermates. Serum cholesterol levels were lower in ABCA1/SR-BI double knockout transplanted animals, as compared to the single knockout and wild-type transplanted animals on Western-type diet. Despite the lower serum cholesterol levels, massive foam cell formation was found in macrophages from spleen and the peritoneal cavity. Interestingly, ABCA1/SR-BI double knockout transplanted animals also showed a major increase in proinflammatory KC (murine interleukin-8) and interleukin-12p40 levels in the circulation. Furthermore, after 10 weeks of Western-type diet feeding, atherosclerotic lesion development in the aortic root was more extensive in the LDLr KO mice reconstituted with ABCA1/SR-BI double knockout bone marrow. CONCLUSIONS: deletion of ABCA1 and SR-BI in bone marrow-derived cells enhances in vivo macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice on Western diet, indicating that under high dietary lipid conditions, both macrophage ABCA1 and SR-BI contribute significantly to cholesterol homeostasis in the macrophage in vivo and are essential for reducing the risk for atherosclerosis.

The scavenger receptor class B, type I is a primary determinant of paraoxonase-1 association with high-density lipoproteins.

OBJECTIVE: To examine the contribution of the scavenger receptor (SR) BI to the mechanism by which high-density lipoprotein (HDL) acquires paraoxonase-1 (PON1). METHODS AND RESULTS: Serum PON1 activity contributes to the antioxidant capacity of HDLs and is suggested to be an independent risk factor for atherosclerosis. The association of PON1 with HDL is a major determinant of its serum activity levels. PON1 secretion was studied in stably transfected Chinese hamster ovary and HepG2 models. Complementary analyses were performed in transgenic models. Modulation of SR-BI expression, by SR-BI small and interfering RNA knockdown and pharmacologically, correlated with significant changes (P<0.01) in PON1 secretion to HDLs and very-low-density lipoproteins. Block lipid transport-1 (BLT1), which increases the affinity of HDL for SR-BI without modulating its expression, was associated with significant increases in secretion. Downregulating postsynaptic density 95/disc-large/zona occludens kinase in HepG2 reduced cell SR-BI protein and lowered enzyme secretion. Serum PON1 activity was significantly reduced in postsynaptic density 95/disc-large/zona occludens kinase knockout mice. CONCLUSIONS: The present study identifies SR-BI as a major determinant of the capacity of HDL to acquire PON1. It reinforces the concept of the receptor as a docking molecule, allowing communication between HDL and the cell, and extends the importance of SR-BI to HDL metabolism and function.

Hepatic glycosphingolipid deficiency and liver function in mice.

UNLABELLED: Recent studies have reported that glycosphingolipids (GSLs) might be involved in obesity-induced insulin resistance. Those reports suggested that inhibition of GSL biosynthesis in animals ameliorated insulin resistance accompanied by improved glycemic control and decreased liver steatosis in obese mice. In addition, pharmacologic GSL depletion altered hepatic secretory function. In those studies, ubiquitously acting inhibitors for GSL biosynthesis have been used to inhibit the enzyme Ugcg (UDP-glucose:ceramide glucosyltransferase), catalyzing the first step of the glucosylceramide-based GSL-synthesis pathway. In the present study a genetic approach for selective GSL deletion in hepatocytes was chosen to achieve complete inhibition of GSL synthesis and to avoid possible adverse effects caused by Ugcg inhibitors. Using the Cre/loxP system under control of the albumin promoter, GSL biosynthesis in hepatocytes and their release into the plasma could be effectively blocked. Deletion of GSL in hepatocytes did not change the quantity of bile excretion through the biliary duct. Total bile salt content in bile, feces, and plasma from mutant mice showed no difference as compared to control animals. Cholesterol concentration in liver, bile, feces, and plasma samples remained unaffected. Lipoprotein concentrations in plasma samples in mutant animals reached similar levels as in their control littermates. No alteration in glucose tolerance after intraperitoneal application of glucose and insulin appeared in mutant animals. A preventive effect of GSL deficiency on development of liver steatosis after a high-fat diet was not observed. CONCLUSION: The data suggest that GSL in hepatocytes are not essential for sterol, glucose, or lipoprotein metabolism and do not prevent high-fat diet-induced liver steatosis, indicating that Ugcg inhibitors exert their effect on hepatocytes either independently of GSL or mediated by other (liver) cell types.

The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2.

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

Lack of Abcg1 results in decreased plasma HDL cholesterol levels and increased biliary cholesterol secretion in mice fed a high cholesterol diet.

OBJECTIVE: The ATP Binding Cassette transporter G1 (ABCG1) has been implicated in cholesterol efflux towards HDL and reverse cholesterol transport (RCT). Biliary cholesterol secretion is considered as an important step in RCT. The aim of the present study was to determine the consequences of Abcg1 deficiency on plasma HDL, liver cholesterol metabolism and biliary cholesterol secretion under conditions of feeding either chow or a 1% cholesterol diet (HCD) or treatment with the LXR agonist T0901317. METHODS AND RESULTS: Abcg1 expression specifically in hepatocytes is induced by both HCD (p<0.01) and T0901317 (p<0.001). HCD or T0901317 treatment resulted in significantly lower plasma HDL cholesterol levels in Abcg1 knockout mice compared with controls (p<0.05) consistent with a role of Abcg1 in cholesterol efflux towards HDL. Liver lipid composition was not affected by the absence of Abcg1. Biliary cholesterol secretion was 47% higher in Abcg1(-/-) mice on HCD (p<0.05) and not different in the chow and the T0901317 groups. The hepatic gene expression profile indicated uniformly throughout the different treatment groups decreased expression of Srebp2 and its target genes HmgCoA reductase (p<0.05) and LDL receptor (p<0.05) in Abcg1(-/-) mice. CONCLUSION: These data demonstrate that Abcg1 (i) contributes to plasma HDL cholesterol levels under conditions of dietary and pharmacological Lxr activation and (ii) might mediate, under conditions of hepatic cholesterol loading, hepatocyte cholesterol efflux towards plasma from a pool accessible for biliary secretion resulting in increased biliary cholesterol output when Abcg1 is lacking.

Independent protective roles for macrophage Abcg1 and Apoe in the atherosclerotic lesion development.

OBJECTIVE: ATP-binding cassette transporter G1 (Abcg1) and apolipoprotein E (Apoe) play a role in macrophage cholesterol efflux and consequently the development of atherosclerosis. A possible interaction between Abcg1 and Apoe in cholesterol efflux was postulated, but the potential combined action of these proteins on atherosclerotic lesion formation is unclear. METHODS: LDL receptor knockout (KO) mice were transplanted with bone marrow from Abcg1/Apoe double KO (dKO) mice, their respective single knockouts, and wild-type (WT) controls and challenged with a high-fat/high-cholesterol diet for 6 weeks to induce atherosclerosis. RESULTS: No differences were found in serum lipid levels. The mean atherosclerotic lesion area in dKO transplanted animals (187+/-18x10(3)microm(2)) was 1.4-fold (p<0.01) increased compared to single knockouts (Abcg1 KO: 138+/-5x10(3)microm(2); Apoe KO: 131+/-7x10(3)microm(2)) and 1.9-fold (p<0.001) as compared to WT controls (97+/-15x10(3)microm(2)). In vitro cholesterol efflux experiments established that combined deletion of Abcg1 and Apoe leads to a larger attenuation of macrophage cholesterol efflux to HDL as compared to single knockouts. CONCLUSIONS: Single deletion of macrophage Abcg1 or Apoe does lead to a moderate non-significant increase in atherosclerotic lesion development as tested by ANOVA, while combined deletion of Abcg1 and Apoe induces a more dramatic and significant increase in atherosclerosis. Our results indicate an additive, independent effect for both macrophage Abcg1 and Apoe in the prevention of atherosclerosis.

Activation of the nuclear receptor PXR decreases plasma LDL-cholesterol levels and induces hepatic steatosis in LDL receptor knockout mice.

To investigate the potential for pregnane X receptor (PXR) ligands as antiatherosclerotic drugs, we have determined the effect of PXR activation on lipid metabolism in an established atherosclerotic mouse model. LDL receptor knockout mice were treated with the PXR agonist PCN. PCN induced a striking 66% decrease in plasma LDL-cholesterol levels. PCN did not affect the cholesterol levels of high-density lipoprotein (HDL) or very-low-density lipoprotein (VLDL). VLDL-triglyceride levels were 2.2-fold increased by PCN, resulting in the presence of triglyceride-rich VLDL particles. This coincided with a 60% decreased hepatic lipase (HL)-mediated plasma lipolysis rate, which could be attributed to a decrease in the hepatic mRNA expression level of both HL (-31%) and its cofactor apolipoprotein A4 (-62%). In the liver, PCN induced a significant increase in the level of triglycerides (+65%) and phospholipids (+72%), a hallmark of hepatic steatosis, leading to a marked increase in Oil red O neutral lipid staining. A similar effect was noticed in ApoE knockout mice. Our studies show that activation of the nuclear receptor PXR by PCN leads to an inhibition of the plasma HL-mediated lipolysis rate, which is associated with a decrease in plasma LDL-cholesterol levels and induction of hepatic steatosis in LDL receptor knockout mice.

Apolipoprotein CI inhibits scavenger receptor BI and increases plasma HDL levels in vivo.

Apolipoprotein CI (apoCI) has been suggested to influence HDL metabolism by activation of LCAT and inhibition of HL and CETP. However, the effect of apoCI on scavenger receptor BI (SR-BI)-mediated uptake of HDL-cholesteryl esters (CE), as well as the net effect of apoCI on HDL metabolism in vivo is unknown. Therefore, we evaluated the effect of apoCI on the SR-BI-mediated uptake of HDL-CE in vitro and determined the net effect of apoCI on HDL metabolism in mice. Enrichment of HDL with apoCI dose-dependently decreased the SR-BI-dependent association of [(3)H]CE-labeled HDL with primary murine hepatocytes, similar to the established SR-BI-inhibitors apoCIII and oxLDL. ApoCI deficiency in mice gene dose-dependently decreased HDL-cholesterol levels. Adenovirus-mediated expression of human apoCI in mice increased HDL levels at a low dose and increased the HDL particle size at higher doses. We conclude that apoCI is a novel inhibitor of SR-BI in vitro and increases HDL levels in vivo.

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI.

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.

Absence of HDL cholesteryl ester uptake in mice via SR-BI impairs an adequate adrenal glucocorticoid-mediated stress response to fasting.

Receptor-mediated cholesterol uptake has been suggested to play a role in maintaining the adrenal intracellular free cholesterol pool and the ability to produce hormones. Therefore, in the current study, we evaluated the importance of scavenger receptor class B type I (SR-BI)-mediated cholesteryl ester uptake from HDL for adrenal glucocorticoid hormone synthesis in vivo. No difference was observed in the plasma level of corticosterone between SR-BI-deficient and wild-type mice under ad libitum feeding conditions. Overnight fasting ( approximately 16 h) stimulated the plasma level of corticosterone by 2-fold in wild-type mice. In contrast, no effect of fasting on plasma corticosterone levels was observed in SR-BI-deficient mice, leading to a 44% lower plasma corticosterone level compared with their wild-type littermate controls. In parallel, an almost complete depletion of lipid stores in the adrenal cortex of fasted SR-BI-deficient mice was observed. Plasma adrenocorticotropic hormone levels were increased by 5-fold in fasted SR-BI-deficient mice. SR-BI deficiency induced marked changes in the hepatic expression of the glucocorticoid-responsive genes cholesterol 7alpha-hydroxylase, HMG-CoA synthase, apolipoprotein A-IV, corticosteroid binding globulin, interleukin-6, and tumor necrosis factor-alpha, which coincided with a 42% decreased plasma glucose level under fasting conditions. In conclusion, we show that the absence of adrenal HDL cholesteryl ester uptake in SR-BI-deficient mice impairs the adrenal glucocorticoid-mediated stress response to fasting as a result of adrenal glucocorticoid insufficiency and attenuated liver glucocorticoid receptor signaling, leading to hypoglycemia under fasting conditions.