Endothelial Zeb2 preserves the hepatic angioarchitecture and protects against liver fibrosis.

AIMS: Hepatic capillaries are lined with specialized liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialization is co-determined by transcription factors. The zinc-finger E-box-binding homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis. METHODS AND RESULTS: To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called 'capillarization'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarization, HSC activation, and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis. CONCLUSION: Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

Unraveling the transcriptional determinants of liver sinusoidal endothelial cell specialization.

Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in blood. LSECs are highly specialized to mediate the clearance of these substances via endocytic scavenger receptors and are equipped with fenestrae that mediate the passage of macromolecules toward hepatocytes. Although some transcription factors (TFs) are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete.Based on a comparison of liver, heart, and brain endothelial cells (ECs), we established a 30-gene LSEC signature comprising both established and newly identified markers, including 7 genes encoding TFs. To evaluate the LSEC TF regulatory network, we artificially increased the expression of the 7 LSEC-specific TFs in human umbilical vein ECs. Although Zinc finger E-box-binding protein 2, homeobox B5, Cut-like homolog 2, and transcription factor EC (TCFEC) had limited contributions, musculoaponeurotic fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and MEIS homeobox 2 (MEIS2) emerged as stronger inducers of LSEC marker expression. Furthermore, a combination of C-MAF, GATA4, and MEIS2 showed a synergistic effect on the increase of LSEC signature genes, including liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN) (or C-type lectin domain family member M (CLEC4M)), mannose receptor C-Type 1 (MRC1), legumain (LGMN), G protein-coupled receptor 182 (GPR182), Plexin C1 (PLXNC1), and solute carrier organic anion transporter family member 2A1 (SLCO2A1). Accordingly, L-SIGN, MRC1, pro-LGMN, GPR182, PLXNC1, and SLCO2A1 protein levels were elevated by this combined overexpression. Although receptor-mediated endocytosis was not significantly induced by the triple TF combination, it enhanced binding to E2, the hepatitis C virus host-binding protein. We conclude that C-MAF, GATA4, and MEIS2 are important transcriptional regulators of the unique LSEC fingerprint and LSEC interaction with viruses. Additional factors are however required to fully recapitulate the molecular, morphological, and functional LSEC fingerprint.NEW & NOTEWORTHY Liver sinusoidal endothelial cells (LSECs) are the first liver cells to encounter waste macromolecules, pathogens, and toxins in the blood and are highly specialized. Although some transcription factors are known to play a role in LSEC specialization, information about the specialized LSEC signature and its transcriptional determinants remains incomplete. Here, we show that Musculoaponeurotic Fibrosarcoma (C-MAF), GATA binding protein 4 (GATA4), and Meis homeobox 2 (MEIS2) are important transcriptional regulators of the unique LSEC signature and that they affect the interaction of LSECs with viruses.

Direct intracerebral delivery of a miR-33 antisense oligonucleotide into mouse brain increases brain ABCA1 expression. [Corrected].

The ATP-binding cassette transporter A1 (ABCA1) is a membrane bound protein that serves to efflux cholesterol and phospholipids onto lipid poor apolipoproteins during HDL biogenesis. Increasing the expression and activity of ABCA1 have beneficial effects in experimental models of various neurologic and cardiovascular diseases including Alzheimer's disease. Despite the beneficial effects of liver X receptor (LXR) agonists--compounds that increase ABCA1 expression--in preclinical studies, their therapeutic utility is limited by systemic adverse effects on lipid metabolism. Interestingly, microRNA-33 (miR-33) inhibition increases ABCA1 expression and activity in rodents and non-human primates without severe metabolic adverse effects. Herein, we demonstrate that treatment of cultured mouse neurons, astrocytes and microglia with an antisense oligonucleotide (ASO) targeting miR-33 increased ABCA1 expression, which was accompanied by increased cholesterol efflux and apoE secretion in astrocytic cultures. We also show that intracerebral delivery of an ASO targeting miR-33 leads to increased ABCA1 expression in cerebral cortex or subcortical structures such as hippocampus. These findings highlight an effective strategy for increasing brain ABCA1 expression/activity for relevant mechanistic studies. [Corrected]

Loss of Cyp8b1 improves glucose homeostasis by increasing GLP-1.

Besides their role in facilitating lipid absorption, bile acids are increasingly being recognized as signaling molecules that activate cell-signaling receptors. Targeted disruption of the sterol 12α-hydroxylase gene (Cyp8b1) results in complete absence of cholic acid (CA) and its derivatives. Here we investigate the effect of Cyp8b1 deletion on glucose homeostasis. Absence of Cyp8b1 results in improved glucose tolerance, insulin sensitivity, and ß-cell function, mediated by absence of CA in Cyp8b1(-/-) mice. In addition, we show that reduced intestinal fat absorption in the absence of biliary CA leads to increased free fatty acids reaching the ileal L cells. This correlates with increased secretion of the incretin hormone GLP-1. GLP-1, in turn, increases the biosynthesis and secretion of insulin from ß-cells, leading to the improved glucose tolerance observed in the Cyp8b1(-/-) mice. Thus, our data elucidate the importance of Cyp8b1 inhibition on the regulation of glucose metabolism.

Hepatic ABCA1 expression improves ß-cell function and glucose tolerance.

Low HDL is a risk factor for the development of type 2 diabetes. Hepatic ABCA1 is the rate-limiting protein in HDL biogenesis, and mice lacking hepatic ABCA1 (ABCA1(-l/-l)) have very low plasma HDL concentrations. To investigate the role of hepatic ABCA1 in glucose tolerance and ß-cell function, we used ABCA1(-l/-l) mice, which showed impaired glucose tolerance without changes in insulin sensitivity. Insulin secretion was reduced following glucose gavage. Ex vivo, glucose stimulated insulin secretion from ß-cells from wild-type (WT) and ABCA1(-l/-l) mice was similar. Insulin secretion was, however, reduced upon addition of ABCA1(-l/-l) serum to the medium compared with WT serum, whereas islets lacking ß-cell ABCA1 were not affected differently by ABCA1(-l/-l) or WT serum. After high-fat feeding, WT and ABCA1(-l/-l) mice showed no difference in glucose tolerance or insulin secretion, and serum from ABCA1(-l/-l) and WT mice fed a high-fat diet did not affect insulin secretion differently. We conclude that hepatic ABCA1 improves glucose tolerance by improving ß-cell function through both HDL production and interaction with ß-cell ABCA1. The beneficial effect of hepatic ABCA1 is decreased under metabolic stress. Increasing hepatic ABCA1 may represent a novel therapeutic strategy for improving glucose homeostasis in diabetes.

ABCA1 in adipocytes regulates adipose tissue lipid content, glucose tolerance, and insulin sensitivity.

Adipose tissue contains one of the largest reservoirs of cholesterol in the body. Adipocyte dysfunction in obesity is associated with intracellular cholesterol accumulation, and alterations in cholesterol homeostasis have been shown to alter glucose metabolism in cultured adipocytes. ABCA1 plays a major role in cholesterol efflux, suggesting a role for ABCA1 in maintaining cholesterol homeostasis in the adipocyte. However, the impact of adipocyte ABCA1 on adipose tissue function and glucose metabolism is unknown. Our aim was to determine the impact of adipocyte ABCA1 on adipocyte lipid metabolism, body weight, and glucose metabolism in vivo. To address this, we used mice lacking ABCA1 specifically in adipocytes (ABCA1(-ad/-ad)). When fed a high-fat, high-cholesterol diet, ABCA1(-ad/-ad) mice showed increased cholesterol and triglyceride stores in adipose tissue, developed enlarged fat pads, and had increased body weight. Associated with these phenotypic changes, we observed significant changes in the expression of genes involved in cholesterol and glucose homeostasis, including ldlr, abcg1, glut-4, adiponectin, and leptin. ABCA1(-ad/-ad) mice also demonstrated impaired glucose tolerance, lower insulin sensitivity, and decreased insulin secretion. We conclude that ABCA1 in adipocytes influences adipocyte lipid metabolism, body weight, and whole-body glucose homeostasis.

Regulation of ABCA1 protein expression and function in hepatic and pancreatic islet cells by miR-145.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) protein maintains cellular cholesterol homeostasis in several different tissues. In the liver, ABCA1 is crucial for high-density lipoprotein biogenesis, and in the pancreas ABCA1 can regulate insulin secretion. In this study, our aim was to identify novel microRNAs that regulate ABCA1 expression in these tissues. APPROACH AND RESULTS: We combined multiple microRNA prediction programs to identify 8 microRNAs that potentially regulate ABCA1. A luciferase reporter assay demonstrated that 5 of these microRNAs (miR-148, miR-27, miR-144, miR-145, and miR-33a/33b) significantly repressed ABCA1 3'-untranslated region activity with miR-145 resulting in one of the larger decreases. In hepatic HepG2 cells, miR-145 can regulate both ABCA1 protein expression levels and cholesterol efflux function. In murine islets, an increase in miR-145 expression decreased ABCA1 protein expression, increased total islet cholesterol levels, and decreased glucose-stimulated insulin secretion. Inhibiting miR-145 produced the opposite effect of increasing ABCA1 protein levels and improving glucose-stimulated insulin secretion. Finally, increased glucose levels in media significantly decreased miR-145 levels in cultured pancreatic beta cells. These findings suggest that miR-145 is involved in glucose homeostasis and is regulated by glucose concentration. CONCLUSIONS: Our studies demonstrate that miR-145 regulates ABCA1 expression and function, and inhibiting this microRNA represents a novel strategy for increasing ABCA1 expression, promoting high-density lipoprotein biogenesis in the liver, and improving glucose-stimulated insulin secretion in islets.

Farnesoid X receptor activation increases cholesteryl ester transfer protein expression in humans and transgenic mice.

Cholesteryl ester transfer protein (CETP) activity results in a proatherogenic lipoprotein profile. In cholestatic conditions, farnesoid X receptor (FXR) signaling by bile acids (BA) is activated and plasma HDL cholesterol (HDL-C) levels are low. This study tested the hypothesis that FXR-mediated induction of CETP contributes to this phenotype. Patients with cholestasis and high plasma BA had lower HDL-C levels and higher plasma CETP activity and mass compared with matched controls with low plasma BA (each P < 0.01). BA feeding in APOE3*Leiden transgenic mice expressing the human CETP transgene controlled by its endogenous promoter increased cholesterol within apoB-containing lipoproteins and decreased HDL-C (each P < 0.01), while hepatic CETP mRNA expression and plasma CETP activity and mass increased (each P < 0.01). In vitro studies confirmed that FXR agonists substantially augmented CETP mRNA expression in hepatocytes and macrophages dependent on functional FXR expression (each P < 0.001). These transcriptional effects are likely mediated by an ER8 FXR response element (FXRE) in the first intron. In conclusion, using a translational approach, this study identifies CETP as novel FXR target gene. By increasing CETP expression, FXR activation leads to a proatherogenic lipoprotein profile. These results have clinical relevance, especially when considering FXR agonists as emerging treatment strategy for metabolic disease and atherosclerosis.

ABCA1 influences neuroinflammation and neuronal death.

ATP-binding cassette transporter A1 (ABCA1) mediates cellular cholesterol efflux in the brain and influences whole brain cholesterol homeostasis. Activation of liver X receptors (LXRs), transcription factors that increase the expression of cholesterol transport genes including ABCA1, reduces neuroinflammation and pathology in neurodegenerative animal models suggesting that in addition to its involvement in cholesterol transport, ABCA1 may play a role in modulating the inflammatory response in the brain. We investigated the cell-type specific role of ABCA1 in neuroinflammation in vivo using mice specifically lacking brain ABCA1 (ABCA1(-B/-B)) as well as mice lacking neuronal (ABCA1(-N/-N)) and astrocytic (ABCA1(-Ast/-Ast)) ABCA1. ABCA1(-B/-B) mice exhibit cortical astrogliosis, increased inflammatory gene expression as well as activation of mitogen-activated protein kinases (MAPKs) following acute lipopolysaccharide (LPS) administration. Microglia cultured from ABCA1(-B/-B) mice exhibit augmented LPS-induced secretion of tumor necrosis factor α (TNFα) and decreased phagocytic activity, indicating an increase in a pro-inflammatory response. ABCA1(-N/-N) mice develop astrogliosis but show no change in inflammatory gene expression. Intriguingly, ABCA1(-Ast/-Ast) mice show neither astrogliosis nor elevated expression of inflammatory markers. Cortical apolipoprotein E (apoE) levels are reduced in ABCA1(-Ast/-Ast) but not in ABCA1(-N/-N) mice, providing in vivo evidence for the specific role of astrocyte ABCA1 in regulating brain apoE levels. Interestingly, cortical neuronal death is increased in 17month-old ABCA1(-B/-B) mice but not in ABCA1(-N/-N) or ABCA1(-Ast/-Ast) mice. Our findings suggest that coordinated ABCA1 activity across neurons and glial cells influences neuroinflammation and neurodegeneration.

Loss of both ABCA1 and ABCG1 results in increased disturbances in islet sterol homeostasis, inflammation, and impaired ß-cell function.

Cellular cholesterol homeostasis is important for normal ß-cell function. Disruption of cholesterol transport by decreased function of the ATP-binding cassette (ABC) transporter ABCA1 results in impaired insulin secretion. Mice lacking ß-cell ABCA1 have increased islet expression of ABCG1, another cholesterol transporter implicated in ß-cell function. To determine whether ABCA1 and ABCG1 have complementary roles in ß-cells, mice lacking ABCG1 and ß-cell ABCA1 were generated and glucose tolerance, islet sterol levels, and ß-cell function were assessed. Lack of both ABCG1 and ß-cell ABCA1 resulted in increased fasting glucose levels and a greater impairment in glucose tolerance compared with either ABCG1 deletion or loss of ABCA1 in ß-cells alone. In addition, glucose-stimulated insulin secretion was decreased and sterol accumulation increased in islets lacking both transporters compared with those isolated from knockout mice with each gene alone. Combined deficiency of ABCA1 and ABCG1 also resulted in significant islet inflammation as indicated by increased expression of interleukin-1ß and macrophage infiltration. Thus, lack of both ABCA1 and ABCG1 induces greater defects in ß-cell function than deficiency of either transporter individually. These data suggest that ABCA1 and ABCG1 each make complimentary and important contributions to ß-cell function by maintaining islet cholesterol homeostasis in vivo.

Restoration of high-density lipoprotein levels by cholesteryl ester transfer protein expression in scavenger receptor class B type I (SR-BI) knockout mice does not normalize pathologies associated with SR-BI deficiency.

OBJECTIVE: Disruption of scavenger receptor class B type I (SR-BI) in mice impairs high-density lipoprotein (HDL)-cholesterol (HDL-C) delivery to the liver and induces susceptibility to atherosclerosis. In this study, it was investigated whether introduction of cholesteryl ester transfer protein (CETP) can normalize HDL-C transport to the liver and reduce atherosclerosis in SR-BI knockout (KO) mice. METHODS AND RESULTS: Expression of human CETP in SR-BI(KO) mice resulted in decreased plasma HDL-C levels, both on chow diet (1.8-fold, P<0.001) and on challenge with Western-type diet (1.6-fold, P<0.01). Furthermore, the presence of CETP partially normalized the abnormally large HDL particles observed in SR-BI(KO) mice. Unexpectedly, expression of CETP in SR-BI(KO) mice did not reduce atherosclerotic lesion development, probably because of consequences of SR-BI deficiency, including the persistence of higher VLDL-cholesterol (VLDL-C) levels, unchanged elevated free cholesterol/total cholesterol ratio, and the increased oxidative status of the animals. In addition, CETP expression did not normalize other characteristics of SR-BI deficiency, including female infertility, reticulocytosis, thrombocytopenia, and impaired platelet aggregation. CONCLUSIONS: CETP restores HDL-C levels in SR-BI(KO) mice, but it does not change the susceptibility to atherosclerosis and other typical characteristics that are associated with SR-BI disruption. This may indicate that the pathophysiology of SR-BI deficiency is not a direct consequence of changes in the HDL pool.

Pioglitazone decreases plasma cholesteryl ester transfer protein mass, associated with a decrease in hepatic triglyceride content, in patients with type 2 diabetes.

OBJECTIVE: Thiazolidinediones reduce hepatic steatosis and increase HDL cholesterol levels. In mice with human-like lipoprotein metabolism (APOE*3-Leiden.CETP transgenic mice), a decrease in hepatic triglyceride content is associated with a decrease in plasma cholesteryl ester transfer protein (CETP) mass and an increase in HDL levels. Therefore, the aim of the present study was to assess the effects of pioglitazone on CETP mass in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: We included 78 men with type 2 diabetes (aged 56.5 +/- 0.6 years; HbA1c 7.1 +/- 0.1%) who were randomly assigned to treatment with pioglitazone (30 mg/day) or metformin (2000 mg/day) and matching placebo, in addition to glimepiride. At baseline and after 24 weeks of treatment plasma HDL cholesterol levels and CETP mass were measured, and hepatic triglyceride content was assessed by proton magnetic resonance spectroscopy. RESULTS Pioglitazone decreased hepatic triglyceride content (5.9 [interquartile range 2.6-17.4] versus 4.1 [1.9-12.3]%, P < 0.05), decreased plasma CETP mass (2.33 +/- 0.10 vs. 2.06 +/- 0.10 microg/ml, P < 0.05), and increased plasma HDL cholesterol level (1.22 +/- 0.05 vs. 1.34 +/- 0.05 mmol/l, P < 0.05). Metformin did not significantly change any of these parameters. CONCLUSIONS: A decrease in hepatic triglyceride content by pioglitazone is accompanied by a decrease in plasma CETP mass and associated with an increase in HDL cholesterol levels. These results in patients with type 2 diabetes fully confirm recent findings in mice.

Bexarotene induces dyslipidemia by increased very low-density lipoprotein production and cholesteryl ester transfer protein-mediated reduction of high-density lipoprotein.

A common dose-limiting side effect of treatment with the retinoid X receptor agonist bexarotene is dyslipidemia. We evaluated the effects of bexarotene on plasma lipid metabolism in patients with metastatic differentiated thyroid carcinoma and investigated the underlying mechanism(s) in apolipoprotein (APO) E*3-Leiden mice without (E3L) and with human cholesteryl ester transfer protein (CETP; E3L.CETP). To this end, 10 patients with metastatic differentiated thyroid carcinoma were treated with bexarotene (300 mg/d) for 6 wk. Bexarotene increased plasma triglyceride (TG; +150%), primarily associated with very low-density lipoprotein (VLDL), and raised plasma total cholesterol (+50%). However, whereas bexarotene increased VLDL-cholesterol (C) and low-density lipoprotein (LDL)-C (+63%), it decreased high-density lipoprotein (HDL)-C (-30%) and tended to decrease apoAI (-18%) concomitant with an increase in endogenous CETP activity (+44%). To evaluate the cause of the bexarotene-induced hypertriglyceridemia and the role of CETP in the bexarotene-induced shift in cholesterol distribution, E3L and E3L.CETP mice were treated with bexarotene through dietary supplementation [0.03% (wt/wt)]. Bexarotene increased VLDL-associated TG in both E3L (+47%) and E3L.CETP (+29%) mice by increasing VLDL-TG production (+68%). Bexarotene did not affect the total cholesterol levels or distribution in E3L mice but increased VLDL-C (+11%) and decreased HDL-C (-56%) as well as apoAI (-31%) in E3L.CETP mice, concomitant with increased endogenous CETP activity (+41%). This increased CETP activity by bexarotene-treatment is likely due to the increase in VLDL-TG, a CETP substrate that drives CETP activity. In conclusion, bexarotene causes combined dyslipidemia as reflected by increased TG, VLDL-C, and LDL-C and decreased HDL-C, which is the result of an increased VLDL-TG production that causes an increase of the endogenous CETP activity.

PXR agonism decreases plasma HDL levels in ApoE3-Leiden.CETP mice.

Pregnane X receptor (PXR) agonism has been shown to affect multiple steps in both the synthesis and catabolism of HDL, but its integrated effect on HDL metabolism in vivo remains unclear. The aim of this study was to evaluate the net effect of PXR agonism on HDL metabolism in ApoE3-Leiden (E3L) and E3L.CETP mice, well-established models for human-like lipoprotein metabolism. Female mice were fed a diet with increasing amounts of the potent PXR agonist 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN). In E3L and E3L.CETP mice, PCN increased liver lipids as well as plasma cholesterol and triglycerides. However, whereas PCN increased cholesterol contained in large HDL-1 particles in E3L mice, it dose-dependently decreased HDL-cholesterol in E3L.CETP mice, indicating that CETP expression dominates the effect of PCN on HDL metabolism. Analysis of the hepatic expression of genes involved in HDL metabolism showed that PCN decreased expression of genes involved in HDL synthesis (Abca1, Apoa1), maturation (Lcat, Pltp) and clearance (Sr-b1). The HDL-increasing effect of PCN, observed in E3L mice, is likely caused by a marked decrease in hepatic SR-BI protein expression, and completely reversed by CETP expression. We conclude that chronic PXR agonism dose-dependently reduces plasma HDL-cholesterol in the presence of CETP.

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.

Niacin increases HDL by reducing hepatic expression and plasma levels of cholesteryl ester transfer protein in APOE*3Leiden.CETP mice.

OBJECTIVE: Niacin potently decreases plasma triglycerides and LDL-cholesterol. In addition, niacin is the most potent HDL-cholesterol-increasing drug used in the clinic. In the present study, we aimed at elucidation of the mechanism underlying its HDL-raising effect. METHODS AND RESULTS: In APOE*3Leiden transgenic mice expressing the human CETP transgene, niacin dose-dependently decreased plasma triglycerides (up to -77%, P<0.001) and total cholesterol (up to -66%, P<0.001). Concomitantly, niacin dose-dependently increased HDL-cholesterol (up to +87%, P<0.001), plasma apoAI (up to +72%, P<0.001), as well as the HDL particle size. In contrast, in APOE*3Leiden mice, not expressing CETP, niacin also decreased total cholesterol and triglycerides but did not increase HDL-cholesterol. In fact, in APOE*3Leiden.CETP mice, niacin dose-dependently decreased the hepatic expression of CETP (up to -88%; P<0.01) as well as plasma CETP mass (up to -45%, P<0.001) and CETP activity (up to -52%, P<0.001). Additionally, niacin dose-dependently decreased the clearance of apoAI from plasma and reduced the uptake of apoAI by the kidneys (up to -90%, P<0.01). CONCLUSIONS: Niacin markedly increases HDL-cholesterol in APOE*3Leiden.CETP mice by reducing CETP activity, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool, and increases HDL-apoAI by decreasing the clearance of apoAI from plasma.

Torcetrapib does not reduce atherosclerosis beyond atorvastatin and induces more proinflammatory lesions than atorvastatin.

BACKGROUND: Although cholesteryl ester transfer protein (CETP) inhibition is regarded as a promising strategy to reduce atherosclerosis by increasing high-density lipoprotein cholesterol, the CETP inhibitor torcetrapib given in addition to atorvastatin had no effect on atherosclerosis and even increased cardiovascular death in the recent Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events trial. Therefore, we evaluated the antiatherogenic potential and adverse effects of torcetrapib in humanized APOE*3-Leiden.CETP (E3L.CETP) mice. METHODS AND RESULTS: E3L.CETP mice were fed a cholesterol-rich diet without drugs or with torcetrapib (12 mg x kg(-1) x d(-1)), atorvastatin (2.8 mg x kg(-1) x d(-1)), or both for 14 weeks. Torcetrapib decreased CETP activity in both the absence and presence of atorvastatin (-74% and -73%, respectively; P<0.001). Torcetrapib decreased plasma cholesterol (-20%; P<0.01), albeit to a lesser extent than atorvastatin (-42%; P<0.001) or the combination of torcetrapib and atorvastatin (-40%; P<0.001). Torcetrapib increased high-density lipoprotein cholesterol in the absence (30%) and presence (34%) of atorvastatin. Torcetrapib and atorvastatin alone reduced atherosclerotic lesion size (-43% and -46%; P<0.05), but combination therapy did not reduce atherosclerosis compared with atorvastatin alone. Remarkably, compared with atorvastatin, torcetrapib enhanced monocyte recruitment and expression of monocyte chemoattractant protein-1 and resulted in lesions of a more inflammatory phenotype, as reflected by an increased macrophage content and reduced collagen content. CONCLUSIONS: CETP inhibition by torcetrapib per se reduces atherosclerotic lesion size but does not enhance the antiatherogenic potential of atorvastatin. However, compared with atorvastatin, torcetrapib introduces lesions of a less stable phenotype.

Atorvastatin increases HDL cholesterol by reducing CETP expression in cholesterol-fed APOE*3-Leiden.CETP mice.

OBJECTIVE: In addition to lowering low-density lipoprotein (LDL)-cholesterol, statins modestly increase high-density lipoprotein (HDL)-cholesterol in humans and decrease cholesteryl ester transfer protein (CETP) mass and activity. Our aim was to determine whether the increase in HDL depends on CETP expression. METHODS AND RESULTS: APOE*3-Leiden (E3L) mice, with a human-like lipoprotein profile and a human-like responsiveness to statin treatment, were crossbred with mice expressing human CETP under control of its natural flanking regions resulting in E3L.CETP mice. E3L and E3L.CETP mice were fed a Western-type diet with or without atorvastatin. Atorvastatin (0.01% in the diet) reduced plasma cholesterol in both E3L and E3L.CETP mice (-26 and -33%, P<0.05), mainly in VLDL, but increased HDL-cholesterol only in E3L.CETP mice (+52%). Hepatic mRNA expression levels of genes involved in HDL metabolism, such as phospholipid transfer protein (Pltp), ATP-binding cassette transporter A1 (Abca1), scavenger receptor class B type I (Sr-b1), and apolipoprotein AI (Apoa1), were not differently affected by atorvastatin in E3L.CETP mice as compared to E3L mice. However, in E3L.CETP mice, atorvastatin down-regulated the hepatic CETP mRNA expression (-57%; P<0.01) as well as the total CETP level (-29%) and cholesteryl esters (CE) transfer activity (-36%; P<0.05) in plasma. CONCLUSIONS: Atorvastatin increases HDL-cholesterol in E3L.CETP mice by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.

The Hyplip2 locus causes hypertriglyceridemia by decreased clearance of triglycerides.

The Hyplip2 congenic mouse strain contains part of chromosome 15 from MRL/MpJ on the BALB/cJ background. Hyplip2 mice show increased plasma levels of cholesterol and predominantly triglycerides (TGs) and are susceptible to diet-induced atherosclerosis. This study aimed at elucidation of the mechanism(s) explaining the hypertriglyceridemia. Hypertriglyceridemia can result from increased intestinal or hepatic TG production and/or by decreased LPL-mediated TG clearance. The intestinal TG absorption and chylomicron formation were studied after intravenous injection of Triton WR1339 and an intragastric load of olive oil containing glycerol tri[(3)H]oleate. No difference was found in intestinal TG absorption. Moreover, the hepatic VLDL-TG production rate and VLDL particle production, after injection of Triton WR1339, were also not affected. To investigate the LPL-mediated TG clearance, mice were injected intravenously with glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles. In Hyplip2 mice, the particles were cleared at a decreased rate (half-life of 25 +/- 6 vs. 11 +/- 2 min; P < 0.05) concomitant with a decreased uptake of emulsion TG-derived (3)H-labeled fatty acids by the liver and white adipose tissue. The increased plasma TG levels in Hyplip2 mice do not result from an enhanced intestinal absorption or increased hepatic VLDL production but are caused by decreased LPL-mediated TG clearance.

Fenofibrate increases HDL-cholesterol by reducing cholesteryl ester transfer protein expression.

In addition to efficiently decreasing VLDL-triglycerides (TGs), fenofibrate increases HDL-cholesterol levels in humans. We investigated whether the fenofibrate-induced increase in HDL-cholesterol is dependent on the expression of the cholesteryl ester transfer protein (CETP). To this end, APOE*3-Leiden (E3L) transgenic mice without and with the human CETP transgene, under the control of its natural regulatory flanking regions, were fed a Western-type diet with or without fenofibrate. Fenofibrate (0.04% in the diet) decreased plasma TG in E3L and E3L.CETP mice (-59% and -60%; P < 0.001), caused by a strong reduction in VLDL. Whereas fenofibrate did not affect HDL-cholesterol in E3L mice, fenofibrate dose-dependently increased HDL-cholesterol in E3L.CETP mice (up to +91%). Fenofibrate did not affect the turnover of HDL-cholesteryl ester (CE), indicating that fenofibrate causes a higher steady-state HDL-cholesterol level without altering the HDL-cholesterol flux through plasma. Analysis of the hepatic gene expression profile showed that fenofibrate did not differentially affect the main players in HDL metabolism in E3L.CETP mice compared with E3L mice. However, in E3L.CETP mice, fenofibrate reduced hepatic CETP mRNA (-72%; P < 0.01) as well as the CE transfer activity in plasma (-73%; P < 0.01). We conclude that fenofibrate increases HDL-cholesterol by reducing the CETP-dependent transfer of cholesterol from HDL to (V)LDL, as related to lower hepatic CETP expression and a reduced plasma (V)LDL pool.