Reduced plasma high-density lipoprotein cholesterol in hyperthyroid mice coincides with decreased hepatic adenosine 5'-triphosphate-binding cassette transporter 1 expression.

The aim of the study was to investigate the influence of severe hyperthyroidism on plasma high-density lipoprotein cholesterol (HDL-C). Recently, it was shown in mice that increasing doses of T(3) up-regulate hepatic expression of scavenger receptor class B, type I, resulting in increased clearance of plasma HDL-C. Here, we show that severe hyperthyroidism in mice did not affect hepatic expression of scavenger receptor class B, type I, but reduced hepatic expression of ATP-binding cassette transporter 1, accompanied by a 40% reduction of HDL-C. The sterol content of bile, liver, and feces was markedly increased, accompanied by up-regulation of hepatic cholesterol 7alpha-hydroxylase, and ATP-binding cassette transporter 5, which is known to promote biliary sterol secretion upon dimerization with ATP-binding cassette transporter 8. Both control and hyperthyroid mice exerted identical plasma clearance of iv injected [(3)H]HDL-C, supporting the view that severe hyperthyroidism does not affect HDL-C clearance but, rather, its formation via hepatic ATP-binding cassette transporter 1.

Phospholipid transfer protein augments apoptosis in THP-1-derived macrophages induced by lipolyzed hypertriglyceridemic plasma.

OBJECTIVE: Lipolysis of triglyceride-rich lipoproteins (TGRLPs) generates phospholipid-rich surface remnants and induces cytotoxic effects in adjacent vascular cells. We hypothesized that by integrating surface remnants into HDL, phospholipid transfer protein (PLTP) alleviates cytotoxicity. METHODS AND RESULTS: To test this hypothesis and gain insight into cytotoxicity during the postprandial phase in vivo, we injected normo-TG and hyper-TG human volunteers after a standardized fat meal (postprandial sample) with heparin, thereby stimulating lipolysis (postprandial heparinized sample). Incubation of (primary) human macrophages and primary human endothelial cells with postprandial heparinized hyper-TG plasma induced pronounced cytotoxic effects that were dose dependent on the TG content of the sample. No such effects were seen with normo-TG and postprandial hyper-TG plasma. In vitro lipolysis of VLDL and chylomicrons indicated that both lipoprotein fractions can cause cytotoxicity. Interestingly, in experiments with THP-1-derived macrophages stably transfected with PLTP, PLTP substantially augmented both net phospholipid uptake and apoptotic cell death due to postprandial heparinized hyper-TG plasma. We observed that activation of caspase-3/7, poly-ADP-ribose polymerase, and enhanced bioactivity of acid sphingomyelinase may all contribute to this augmented apoptosis. CONCLUSIONS: Our data show that lipolysis of TGRLPs and their remodelling by PLTP interact to disturb cellular phospholipid flux and intracellular signaling processes, ultimately leading to apoptosis in human macrophages and endothelial cells.

Aspirin regulates expression and function of scavenger receptor-BI in macrophages: studies in primary human macrophages and in mice.

Scavenger receptor class B type I (SR-BI) has been shown to be expressed in human atherosclerotic plaque macrophages, where it is believed to reduce atherosclerosis by promoting cholesterol efflux. In this study we investigated the influence of aspirin and other NSAIDs on SR-BI expression and function in cultured human macrophages as well as in different mouse strains. Incubation of human macrophages with 0.5 mmol/l aspirin resulted in increased SR-BI protein expression and increased uptake of HDL-associated [3H]cholesteryl oleate without changes of SR-BI mRNA levels. In contrast, using 5 mmol/l of aspirin, SR-BI expression and function were significantly decreased. Sodium salicylate exerted similar effects on SR-BI expression, whereas no effects were observed using known COX1/2 inhibitors ibuprofen and naproxen, respectively. In in vivo studies low-dose aspirin treatment (6 mg/kg.day) induced SR-BI expression in wild-type and PPAR-alpha knockout mice, respectively, whereas the opposite effect was observed upon high-dose aspirin treatment (60 mg/kg.day) in these animals. We could show that COX-independent effects of aspirin were able to enhance expression of SR-BI in macrophages in a post-transcriptional, PPAR-alpha independent way, suggesting a novel pharmacologic effect of aspirin.

Scavenger receptor class B type I polymorphisms and peripheral arterial disease.

Genetic variations of the scavenger receptor class B type I (SR-BI) have been demonstrated to be associated with plasma lipid parameters, anthropomorphic parameters, and coronary artery disease. We determined the frequency of 3 single-nucleotide polymorphisms within the SR-BI gene (SCARB1) in 354 patients with peripheral arterial disease (PAD) and 354 controls matched for age, sex, and diabetes and related to lipids and disease state, that is, PAD. SCARB1 combined genotype exon 1/intron 5/exon 8 were found to be associated with plasma total and low-density lipoprotein cholesterol levels, respectively. In terms of disease, a significant risk for PAD was observed in female subjects carrying the common allele of exon 8 (odds ratio, 2.623; 95% confidence interval, 1.321-5.208; P=.003). The variant allele of intron 5 was found to be a risk factor for PAD in men (odds ratio, 2.182; 95% confidence interval, 1.288-3.698; P=.005). Furthermore, the SCARB1 combined genotype intron 5/exon 8 proved predictive for PAD in the whole population (P=.006), which remained significant after correction for traditional risk factors. In conclusion, in the present study population, SCARB1 polymorphisms not only show associations with plasma levels of total and low-density lipoprotein cholesterol, respectively, but also with the risk for PAD.

Increased plasma levels of LDL cholesterol in rabbits after adenoviral overexpression of human scavenger receptor class B type I.

Scavenger receptor class B type I (SR-BI), a CD36 family member, plays a key role in high-density lipoprotein (HDL) metabolism, reverse cholesterol transport, and whole body cholesterol homeostasis, and is shown to be involved in the development of atherosclerosis in mice. In this report, we describe the effects of the adenoviral overexpression of human SR-BI (hSR-BI) in New Zealand White (NZW) rabbits, a wild-type animal model that expresses cholesteryl ester transfer protein (CETP) in plasma, displays a manlike lipoprotein profile, and is susceptible to atherosclerosis. A total of 1x10(12) adenoviral particles containing either hSR-BI or lacZ complementary deoxyribonucleic acid (control) were infused into the ear vein of NZW rabbits. Transgene expression was ascertained by TaqMan Real Time polymerase chain reaction measurements. Rabbits infected with Ad/hSR-BI (adenoviral plasmids containing hSR-BI) showed a faster clearance of administered [3H]HDL cholesterol and significantly decreased apolipoprotein (apo) A-I levels when compared to control rabbits, respectively. Interestingly, we found markedly increased levels of low-density lipoprotein (LDL) cholesterol exclusively in SR-BI-overexpressing rabbits. These changes were not accompanied by alterations in LDL receptor expression but by increased levels of CE transfer in these animals. By lowering HDL cholesterol and increasing plasma apoB-containing lipoprotein levels, the overexpression of SR-BI leads to a lipoprotein pattern, which is believed to enhance the development of atherosclerosis. The role of SR-BI in lipoprotein metabolism and atherogenesis in rabbits--a CETP-expressing animal model displaying a manlike lipoprotein profile--may therefore be different from the one found in rodents.

In vivo application of adenoviral vectors purified by a Taqman Real Time PCR-supported chromatographic protocol.

Recombinant adenoviral vectors encoding human HDL-cholesterol receptor SR-BI (Ad/hSR-BI) or beta-galactosidase (Ad/lacZ), respectively, were purified using a Source15 Q anion-exchange (AEX) column and quantified using two parallel Taqman Real Time PCR systems with different target sequences. Adenovirus concentrations were ascertained by 260 nm measurements, purity by 260/280 nm ratio and SDS-PAGE. Subsequently, adenoviruses were validated by experimental intravenous application into New Zealand White rabbits. Transgene expression was verified by functional assays determining plasma clearance rate of 3H-HDL-cholesterol, and was not affected by 21-months storage at -80 degrees C. No alterations of liver enzymes and C-reactive protein (CRP) upon Source15 Q adenovirus treatment could be detected, demonstrating biological safety of our protocol.

The liver-selective thyromimetic T-0681 influences reverse cholesterol transport and atherosclerosis development in mice.

BACKGROUND: Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls. CONCLUSIONS/SIGNIFICANCE: The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans.

Influence of aspirin on SR-BI expression in human carotid plaques.

BACKGROUND: We recently showed that aspirin promotes scavenger receptor class-B type I (SR-BI) protein expression in vitro in primary human macrophages and in vivo in resident peritoneal macrophages of mice. METHODS: We compared SR-BI and CD68 expression in carotid atherosclerotic specimens from endarterectomized patients with (n=38) or without (n=19) low-dose aspirin medication (100 mg/day) prior to endarterectomy. RESULTS: We found no differences concerning expression of CD68, indicating that aspirin did not influence macrophage content within atherosclerotic plaques. However, aspirin increased the expression of SR-BI protein in the analyzed specimens. In human THP-1-derived macrophages, induction of SR-BI protein by aspirin was abrogated by concomitant pharmacological inhibition of nuclear factor-kappa B (NF-kappaB). In in vitro experiments employing cultured primary macrophages from NF-kappaB/p50 KO mice, aspirin was not able to influence SR-BI expression. Additionally, no considerable effects on SR-BI expression were observed in vivo in resident macrophages of NF-kappaB/p50 KO mice orally treated with low or high doses of aspirin, respectively. CONCLUSIONS: We suggest that aspirin treatment might lead to enhanced expression of SR-BI in human plaque macrophages and that this effect is dependent on the presence of NF-kappaB.

The thyromimetic T-0681 protects from atherosclerosis.

This report describes studies in hyperlipidemic New Zealand White (NZW) rabbits investigating the impact of the liver-selective thyromimetic T-0681 on lipoprotein metabolism and the development of atherosclerosis. Prolonged treatment with T-0681 increased the hepatic expression of both LDL receptor and scavenger receptor class B, type I without affecting cholesteryl ester transfer protein activity. Upregulation of hepatic lipoprotein receptors was accompanied by a marked decrease of apolipoprotein B-containing lipoproteins, reflected by a 60% reduction of plasma cholesterol and a >70% reduction of plasma triglyceride levels. Most importantly, T-0681 reduced the development of atherosclerosis by 80% in NZW rabbits on high-cholesterol chow. Our data suggest that liver-selective thyromimetics, such as T-0681, may prove to be useful therapeutic agents against the development of atherosclerosis in humans.

The MTP -493TT genotype is associated with peripheral arterial disease: results from the Linz Peripheral Arterial Disease (LIPAD) Study.

OBJECTIVES: Microsomal triglyceride transfer protein (MTP) transfers lipids into apoprotein B-containing lipoproteins for secretion from liver, intestine, and heart. We hypothesized the -493T single nucleotide polymorphism in the MTP promoter region to be associated with altered lipoprotein levels and with presence of peripheral arterial disease (PAD). DESIGN AND METHODS: 433 patients with symptomatic PAD and 433 controls matched for sex and age from the Linz Peripheral Arterial Disease (LIPAD) study were genotyped cross-sectionally for the -493T single nucleotide polymorphism in the promoter region of the MTP gene. RESULTS: The frequency of the -493T allele in patients with PAD was 0.320, whereas it was 0.255 in controls (p<0.001). The MTP -493TT genotype was independently associated with PAD, even after adjustment for LDL cholesterol. The odds ratio of the -493TT MTP genotype for PAD was 3.18 (95% CI, 1.76-5.71) when adjusted for current smoking, arterial hypertension, LDL cholesterol, triglycerides, glycohemoglobin, C-reactive protein, and homocysteine. Furthermore, we found an association between the MTP promoter polymorphism and the apolipoprotein B-containing lipoproteins total-cholesterol (p=0.011), LDL cholesterol (p=0.002) and apolipoprotein B (p=0.034). CONCLUSIONS: Our results provide preliminary evidence for a potential role of the MTP -493TT genotype in the pathogenesis of PAD.

Low phospholipid transfer protein (PLTP) is a risk factor for peripheral atherosclerosis.

OBJECTIVE: Phospholipid transfer protein (PLTP) facilitates cholesterol efflux from cells, intravascular HDL remodelling and transfer of vitamin E and endotoxin. In humans, the relationship of PLTP to atherosclerosis is unknown. However, strong coronary risk factors like obesity, diabetes, cigarette smoking and inflammation increase circulating levels of active PLTP. The aim of the present, cross-sectional study was to analyze the relationship of PLTP to peripheral arterial disease, a marker of generalized atherosclerosis, independently of potentially confounding factors like obesity, diabetes and smoking. METHODS: We performed a case control study in 153 patients with symptomatic peripheral arterial disease (PAD) and 208 controls free of vascular disease. Smokers and patients with diabetes mellitus were excluded. A lipoprotein-independent assay was used for measurement of circulating bioactive PLTP and an ELISA utilizing a monoclonal antibody was used to analyze PLTP mass. RESULTS: PLTP activity was significantly decreased in patients with PAD 5.5 (4.6-6.4)(median (25th-75th percentile)) versus 5.9 (5.1-6.9) micromol/mL/h in controls (p=0.001). In contrast, PLTP mass was similar in patients with PAD 8.5 microg/mL (7.3-9.5) and in controls 8.3 microg/mL (6.9-9.7) (p=0.665). Multivariate logistic regression analysis revealed that PLTP activity is independently associated with the presence of PAD. PLTP activity was similar in patients with and without lipid-lowering drugs (p=0.396). CONCLUSION: Our results show that in non-diabetic, non-smoking subjects low rather than high PLTP activity is a marker for the presence of peripheral arterial disease and that distribution of PLTP between high-activity and low-activity forms may be compromised in atherosclerosis.

Hepatic ENPP1 expression is induced in diabetic rabbits.

The ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an inhibitor of the insulin receptor. Variants of ENPP1 are associated with infantile arterial calcification, obesity, and insulin resistance. To study the functional relevance of this protein in vivo, we cloned rabbit ENPP1 and studied its regulation in experimentally induced diabetes mellitus. We amplified and sequenced the complete coding sequence of rabbit ENPP1 gene out of a liver cDNA library using redundant primers deduced from other species. Next, we performed quantitative PCR of ENPP1 to study the tissue distribution of ENPP1 expression and its regulation in an alloxan-dependent diabetes model. The putative rabbit ENPP1 protein contains 873 amino acids and is highly conserved when compared with human ENPP1 (90% amino acid identity). Particularly high levels of ENPP1 mRNA expression were found in adipose tissue. Quantitative PCR analysis revealed a significant upregulation of ENPP1 transcription in liver (p = 0.025) and brain (p = 0.034) of diabetic rabbits compared with controls. Hepatic ENPP1 expression is induced in diabetic rabbits when compared with controls. This approximately twofold upregulation of ENPP1 mRNA in rabbit liver parallels previous findings in patients with type 2 diabetes mellitus. We provide further molecular information about ENPP1 as a potential pharmacologic target and characterize its regulation in an insulin-dependent diabetes mellitus animal model.