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Abstract: 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 (100mg/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-κB). In in vitro experiments employing cultured primary macrophages from NF-κB/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-κB/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-κB. [Copyright &y& Elsevier]

Abstract: Background: Determination of the diagnostic usefulness of proton transfer reaction mass spectrometry (PTR-MS) for detecting primary lung cancer through analysis of volatile organic compounds (VOCs) in exhaled human breath was demonstrated in this investigation. Unlike, for example, gas-chromatographic analyses, PTR-MS can be used without time-consuming preconcentration of the gas samples. Methods: By means of PTR-MS, exhaled breath samples from primary lung cancer patients (n =17) were analyzed and compared with both an overall control collective (controls total, n =170) and three sub-collectives: hospital personnel (controls hospital, n =35), age-matched persons (controls age, n =25), and smokers (controls s, n =60), respectively. Results: Among the VOCs present at reasonably high concentrations, the ones leading to the product ion at m/z =31 (VOC-31, tentatively protonated formaldehyde) and m/z =43 (VOC-43, tentatively a fragment of protonated iso-propanol), were found at significantly higher concentrations in the breath gas of the primary lung cancer patients as compared to the healthy controls at the following median concentrations (with interquartile distance, iqr): For VOC-31 the median concentrations were 7.0ppb (iqr, 15.5ppb) versus 3.0ppb (iqr, 1.9ppb) with P <10−4. For VOC-43 the median concentrations were 244.1ppb (iqr, 236.2ppb) versus 94.1ppb (iqr, 55.2ppb) with P <10−6. The discriminative power between the two collectives was further assessed by ROC-curves obtained upon variation of the chosen threshold concentration and by Fisher''s Quadratic Discriminant Method. Conclusions: Within the limits of pilot study, VOC-31 and -43 were found to best discriminate between exhaled breath of primary lung cancer cases and healthy controls. Simple and time-saving breath gas analysis by PTR-MS makes this method attractive for a larger clinical evaluation. It may become a new valuable tool for diagnosing primary lung cancer. [Copyright &y& Elsevier]

Abstract: Recombinant adenoviral vectors encoding human HDL-cholesterol receptor SR-BI (Ad/hSR-BI) or β-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 260nm measurements, purity by 260/280nm 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°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. [Copyright &y& Elsevier]

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-α 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-α independent way, suggesting a novel pharmacologic effect of aspirin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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)μmol/mL/h in controls (p =0.001). In contrast, PLTP mass was similar in patients with PAD 8.5μg/mL (7.3–9.5) and in controls 8.3μg/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. [Copyright &y& Elsevier]

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 1×1012 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. [ABSTRACT FROM AUTHOR]