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.

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.).

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.

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.

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.

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.

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.

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 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.

Coexistence of foam cells and hypocholesterolemia in mice lacking the ABC transporters A1 and G1.

The concept that macrophages can become foam cells as a result of a disturbed balance between the uptake of cholesterol from lipoproteins and cholesterol efflux is generally accepted. ABCA1 and ABCG1 are two cholesterol transporters that may act sequentially to remove cellular cholesterol, but currently their combined role in vivo is unknown. We report here that targeted disruption of both ABCA1 and ABCG1 in mice, despite severe plasma hypocholesterolemia, leads to massive lipid accumulation and foam cell formation of tissue macrophages. A complete ablation of cellular cholesterol efflux in vitro is observed, whereas in vivo macrophage-specific reverse cholesterol transport to the feces is markedly decreased. Despite the massive foam cell formation of tissue macrophages, no lipid accumulation was observed in the vascular wall, even in mice of 1 year old, indicating that the double knockout mice, possibly because of their hypocholesterolemia, lack the trigger to attract macrophages to the vessel wall. In conclusion, even under hypocholesterolemic conditions macrophages can be converted into foam cells, and ABCA1 and ABCG1 play an essential role in the prevention of foam cell formation.

Combined deletion of macrophage ABCA1 and ABCG1 leads to massive lipid accumulation in tissue macrophages and distinct atherosclerosis at relatively low plasma cholesterol levels.

OBJECTIVE: The purpose of this study was to evaluate the effect of the combined deletion of ABCA1 and ABCG1 expression in macrophages on foam cell formation and atherosclerosis. METHODS AND RESULTS: LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/ABCG1 double KO (dKO) mice. Plasma cholesterol levels after 6 weeks of Western-type diet (WTD) feeding were significantly lower in dKO transplanted mice than ABCA1 KO, ABCG1 KO, and control transplanted animals. Extreme foam cell formation was present in macrophages of various tissues and the peritoneal cavity of dKO transplanted animals. Furthermore, severe hypoplasia of the thymus and a significant decrease in CD4-positive T cells in blood was observed. Despite relatively low plasma cholesterol levels dKO transplanted animals developed lesion sizes of 156+/-19x10(3) microm2 after only 6 weeks of WTD feeding. Lesions, however, were smaller than single ABCA1 KO transplanted animals (226+/-30x10(3) microm2; P<0.05) and not significantly different from single ABCG1 KO (117+/-22x10(3) microm2) and WT transplanted mice (112+/-15x10(3) microm2). CONCLUSIONS: Macrophage ABCA1 and ABCG1 play a crucial role in the prevention of macrophage foam cell formation, whereas combined deletion only modestly influences atherosclerosis which is associated with an attenuated increase in WTD-induced plasma cholesterol and decreased proinflammatory CD4-positive T cell counts.

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.

Total body ABCG1 expression protects against early atherosclerotic lesion development in mice.

OBJECTIVE: ABCG1 has recently been identified as a facilitator of cholesterol and phospholipid efflux from macrophages to HDL. In bone marrow transplantation studies, we and others have now shown that the absence of macrophage ABCG1 may differentially influence atherosclerotic lesions dependent on the experimental setting and/or the stage of atherosclerotic lesion development. To further define the role of ABCG1 in atherogenesis, we investigated in the current study the effect of total body deficiency of ABCG1 on atherosclerotic lesion development. METHODS AND RESULTS: ABCG1-/- mice and wild-type littermates were fed an atherogenic diet for 12 weeks to induce atherosclerotic lesion formation. Both before and after the start of the atherogenic diet, serum lipid levels and lipoprotein profiles did not differ significantly between the two groups. In addition no significant difference in serum apoE levels was found after diet feeding. In wild-type mice the atherogenic diet induced the formation of macrophage-rich early lesions (size: 24+/-7x10(3) microm2 [n=6]). Feeding ABCG1-/- mice the atherogenic diet led to a significant 1.9-fold stimulation of atherosclerotic lesion size (46+/-6x10(3) microm2 [n=7]; Student t test P=0.034 and Mann-Whitney test P=0.050) compared with controls, suggesting a clear antiatherogenic role for ABCG1. At the same time, excessive lipid accumulation was observed in macrophage-rich areas of the lungs and spleens of ABCG1-/- mice as compared with wild-type mice. CONCLUSIONS: Total body ABCG1 expression protects against early atherosclerotic lesion development.

Platelet activation by oxidized low density lipoprotein is mediated by CD36 and scavenger receptor-A.

OBJECTIVE: The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2' (apoER2')-mediated signaling to p38(MAPK) and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca2+. Here we report a new mechanism for platelet activation by oxLDL. METHODS AND RESULTS: Oxidation of nLDL increases p38(MAPK) activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptor-associated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38(MAPK) activation by oxLDL but combined blockade inhibits p38(MAPK) by >40% and platelet adhesion to fibrinogen under flow by >60%. Mouse platelets deficient in either CD36 or SR-A show normal p38(MAPK) activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38(MAPK) by >70%. CONCLUSION: These findings reveal a novel platelet-activating pathway stimulated by oxLDL that is initiated by the combined action of CD36 and SR-A.

The pharmacokinetic behavior of the photosensitizer meso-tetra-hydroxyphenyl-chlorin in mice and men.

PURPOSE: Meso-tetra-hydroxyphenyl-chlorin (mTHPC) is a hydrophobic photosensitizer that binds to plasma lipoproteins after intravenous injection. In vitro experiments with human plasma have shown that mTHPC initially binds to an unknown protein and subsequently redistributes to lipoprotein fractions. It has been suggested that this might explain the unusual pharmacokinetic profile of mTHPC humans. In humans, unlike in rodents, reappearance of mTHPC has been reported, resulting in a second plasma peak after intravenous injection. However, previous studies analyzed only limited time points during the first 24 h after injection. Our aim was to determine the pharmacokinetics of mTHPC in detail, and to investigate whether the pharmacokinetic behavior of the drug is affected by binding of mTHPC to lipoproteins in vivo. METHODS: Plasma of cancer patients and mice, intravenously injected with mTHPC, was analyzed for total drug content and drug distribution over the lipoprotein fractions. RESULTS: Pharmacokinetic profiles of mTHPC in a group of human subjects showed that apparent steady state drug levels were maintained for at least 10 h. Closer examination of individual profiles showed that the initial (5 min) plasma drug levels were on average 86% of the maximal plasma concentration, which occurred at about 5 h after injection. In mice, however, plasma pharmacokinetics were described by a standard bi-exponential decline of the drug concentration. The majority (>58%) of mTHPC injected into both BALB/c nude mice and patients initially bound to the HDL plasma fraction. We extended our study to ApoE -/- mice, with highly elevated lipoprotein levels, and SR-BI -/- mice, which are lacking the main clearance pathway for HDL associated cholesteryl esters, to take into account the differences between lipoprotein levels and clearance in mice and man. Although mTHPC distribution over the lipoproteins changed in these mice, pharmacokinetic profiles of mTHPC remained the same. CONCLUSIONS: We conclude that neither lipoprotein levels nor cholesterol metabolism affects the pharmacokinetics of mTHPC in plasma.

Macrophage ABCG1 deletion disrupts lipid homeostasis in alveolar macrophages and moderately influences atherosclerotic lesion development in LDL receptor-deficient mice.

OBJECTIVE: ABCG1 has recently been identified as a facilitator of cellular cholesterol and phospholipid efflux to high-density lipoprotein (HDL). Its expression in macrophages is induced during cholesterol uptake in macrophages and by liver X receptor (LXR). The role of macrophage ABCG1 in atherosclerotic lesion development is, however, still unknown. METHODS AND RESULTS: To assess the role of macrophage ABCG1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr-/-) mice that are selectively deficient in macrophage ABCG1 by using bone marrow transfer (ABCG1-/- --> LDLr-/-). Peritoneal macrophages isolated from donor ABCG1-/- mice exhibited a 22% (P=0.0007) decrease in cholesterol efflux to HDL. To induce atherosclerosis, transplanted mice were fed a high-cholesterol diet containing 0.25% cholesterol and 15% fat for 6 and 12 weeks. Serum lipid levels and lipoprotein profiles did not differ significantly between ABCG1-/- --> LDLr-/- mice and controls. In lungs of ABCG1-/- --> LDLr-/- mice a striking accumulation of lipids was observed in macrophages localized to the subpleural region. After 6 weeks of high-cholesterol diet feeding the atherosclerotic lesion size was 49+/-12x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 65+/-15x103 microm2 for ABCG1-/- --> LDLr-/- mice and after 12 weeks of high-cholesterol diet feeding 124+/-17x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 168+/-17x10(3) microm2 for ABCG1-/- --> LDLr-/- mice. Atherosclerotic lesion size depended on both time and the macrophage ABCG1 genotype (P=0.038 by 2-way ANOVA, n > or = 8), indicating a moderately 33% to 36% increase in lesion formation in the absence of macrophage ABCG1. CONCLUSIONS: Macrophage ABCG1 deficiency does lead to heavy lipid accumulation in macrophages of the lung, and also a moderately significant effect on atherosclerotic lesion development was observed.

Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development.

Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Macrophages cannot limit the uptake of cholesterol and therefore depend on cholesterol efflux pathways for preventing their transformation into foam cells. Several ABC-transporters, including ABCA1 and ABCG1, facilitate the efflux of cholesterol from macrophages. These transporters, however, also affect membrane lipid asymmetry which may have important implications for cellular endocytotic pathways. We propose that in addition to the generally accepted role of these ABC-transporters in the prevention of foam cell formation by induction of cholesterol efflux from macrophages, they also influence the macrophage endocytotic uptake.

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.

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.