Cholesterol Transport In Mice Research Articles

Danlou Recipe promotes cholesterol efflux in macrophages RAW264.7 and reverses cholesterol transport in mice with hyperlipidemia induced by P407

IntroductionLiver X Receptor (LXR) agonists could attenuate the development of atherosclerosis but bring excess lipid accumulation in the liver. Danlou Recipe was believed to be a benefit for improving the lipid profile. Thus, it is unclear whether Danlou Recipe could attenuate hyperlipidemia without excess lipid accumulated in the liver of mice. This study aimed to clarify if Danlou Recipe could alleviate the progression of hyperlipidemia in mice without extra lipids accumulated in the liver.MethodsMale murine macrophage RAW264.7 cells and murine peritoneal macrophages were used for the in vitro experiments. Cellular cholesterol efflux was determined using the fluorescent cholesterol labeling method. Those genes involved in lipid metabolism were evaluated by qRT‐PCR and western blotting respectively. In vivo, a mouse model of hyperlipidemia induced by P407 was used to figure out the effect of Danlou Recipe on reverse cholesterol transport (RCT) and hyperlipidemia. Ethanol extract of Danlou tablet (EEDL) was prepared by extracting the whole powder of Danlou Prescription from ethanol, and the chemical composition was analyzed by ultra-performance liquid chromatography (UPLC).ResultsEEDL inhibits the formation of RAW264.7 macrophage-derived foam cells, and promotes ABCA1/apoA1 conducted cholesterol efflux in RAW264.7 macrophages and mouse peritoneal macrophages. In the P407-induced hyperlipidemia mouse model, oral administration of EEDL can promote RCT in vivo and improve fatty liver induced by a high-fat diet. Consistent with the findings in vitro, EEDL promotes RCT by upregulating the LXR activities.ConclusionOur results demonstrate that EEDL has the potential for targeting RCT/LXR in the treatment of lipid metabolism disorders to be developed as a safe and effective therapy.

A Timely Review of Cross-Kingdom Regulation of Plant-Derived MicroRNAs.

MicroRNAs (miRNAs) belong to a class of non-coding RNAs that suppress gene expression by complementary oligonucleotide binding to the sites in target messenger RNAs. Numerous studies have demonstrated that miRNAs play crucial role in virtually all cellular processes of both plants and animals, such as cell growth, cell division, differentiation, proliferation and apoptosis. The study of rice MIR168a has demonstrated for the first time that exogenous plant MIR168a influences cholesterol transport in mice by inhibiting low-density lipoprotein receptor adapter protein 1 expression. Inspired by this finding, the cross-kingdom regulation of plant-derived miRNAs has drawn a lot of attention because of its capability to provide novel therapeutic agents in the treatment of miRNA deregulation-related diseases. Notably, unlike mRNA, some plant miRNAs are robust because of their 3′ end modification, high G, C content, and the protection by microvesicles, miRNAs protein cofactors or plant ingredients. The stability of these small molecules guarantees the reliability of plant miRNAs in clinical application. Although the function of endogenous miRNAs has been widely investigated, the cross-kingdom regulation of plant-derived miRNAs is still in its infancy. Herein, this review summarizes the current knowledge regarding the anti-virus, anti-tumor, anti-inflammatory, anti-apoptosis, immune modulation, and intestinal function regulation effects of plant-derived miRNAs in mammals. It is expected that exploring the versatile role of plant-derived miRNAs may lay the foundation for further study and application of these newly recognized, non-toxic, and inexpensive plant active ingredients.

Endothelial Lipase Is Involved in Cold-Induced High-Density Lipoprotein Turnover and Reverse Cholesterol Transport in Mice.

The physiologic activation of thermogenic brown and white adipose tissues (BAT/WAT) by cold exposure triggers heat production by adaptive thermogenesis, a process known to ameliorate hyperlipidemia and protect from atherosclerosis. Mechanistically, it has been shown that thermogenic activation increases lipoprotein lipase (LPL)-dependent hydrolysis of triglyceride-rich lipoproteins (TRL) and accelerates the generation of cholesterol-enriched remnants and high-density lipoprotein (HDL), which promotes cholesterol flux from the periphery to the liver. HDL is also subjected to hydrolysis by endothelial lipase (EL) (encoded by LIPG). Genome-wide association studies have identified various variants of EL that are associated with altered HDL cholesterol levels. However, a potential role of EL in BAT-mediated HDL metabolism has not been investigated so far. In the present study, we show that in mice, cold-stimulated activation of thermogenic adipocytes induced expression of Lipg in BAT and inguinal WAT but that loss of Lipg did not affect gene expression of thermogenic markers. Furthermore, in both wild type (WT) and Lipg-deficient mice, activation of thermogenesis resulted in a decline of HDL cholesterol levels. However, cold-induced remodeling of the HDL lipid composition was different between WT and Lipg-deficient mice. Notably, radioactive tracer studies with double-labeled HDL indicated that cold-induced hepatic HDL cholesterol clearance was lower in Lipg-deficient mice. Moreover, this reduced clearance was associated with impaired macrophage-to-feces cholesterol transport. Overall, these data indicate that EL is a determinant of HDL lipid composition, cholesterol flux, and HDL turnover in conditions of high thermogenic activity.

The Role of Phospholipid Transfer Protein in the Development of Atherosclerosis.

Phospholipid transfer protein (PLTP), a member of lipid transfer protein family, is an important protein involved in lipid metabolism in the circulation. This article reviews recent PLTP research progresses, involving lipoprotein metabolism and atherogenesis. PLTP activity influences atherogenic and anti-atherogenic lipoprotein levels. Human serum PLTP activity is a risk factor for human cardiovascular disease and is an independent predictor of all-cause mortality. PLTP deficiency reduces VLDL and LDL levels and attenuates atherosclerosis in mouse models, while PLTP overexpression exerts an opposite effect. Both PLTP deficiency and overexpression result in reduction of HDL which has different size, inflammatory index, and lipid composition. Moreover, although both PLTP deficiency and overexpression reduce cholesterol efflux capacity, but this effect has no impact in macrophage reverse cholesterol transport in mice. Furthermore, PLTP activity is related with metabolic syndrome, thrombosis, and inflammation. PLTP could be target for the treatment of dyslipidemia and atherosclerosis, although some potential off-target effects should be noted.

Antisense oligonucleotide–mediated inhibition of angiopoietin-like protein 3 increases reverse cholesterol transport in mice

Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers. The effect of ANGPTL3 on HDL-C is typically attributed to its function as an inhibitor of the enzyme endothelial lipase. The ability to facilitate reverse cholesterol transport (RCT), the transport of cholesterol from peripheral tissues back to the liver, is a proposed antiatherogenic property of HDL. However, the effect of ANGPTL3 inhibition on RCT remains unclear. Here, we performed a series of dose-response and RCT studies using an Angptl3 antisense oligonucleotide (ASO) in mouse models with varying plasma lipid profiles ranging from moderately to severely hyperlipidemic. Angptl3 ASO-mediated reduction in HDL-C was limited to the model with moderate lipidemia, where the majority of plasma cholesterol was associated with HDL. Surprisingly, regardless of the effect on HDL-C, treatment with the Angptl3 ASO enhanced RCT in all models tested. The observations from the RCT assays were confirmed in HDL clearance studies, where mice treated with the Angptl3 ASO displayed increased plasma clearance and hepatic uptake of labeled HDL. The results from our studies suggest that inhibition of ANGPTL3 not only reduces levels of proatherogenic lipids but also improves HDL-mediated RCT.

Hepatic Overexpression of Endothelial Lipase Lowers High-Density Lipoprotein but Maintains Reverse Cholesterol Transport in Mice: Role of Scavenger Receptor Class B Type I/ATP-Binding Cassette Transporter A1-Dependent Pathways.

Reverse cholesterol transport (RCT) is a major mechanism by which HDL (high-density lipoprotein) protects against atherosclerosis. Endothelial lipase (EL) reportedly reduces HDL levels, which, in theory, would increase atherosclerosis. However, it remains unclear whether EL affects RCT in vivo. Adenoviral vectors expressing EL or luciferase were intravenously injected into mice, and a macrophage RCT assay was performed. As expected, hepatic EL overexpression markedly reduced HDL levels. In parallel, plasma 3H-cholesterol counts from the EL-expressing mice decreased by 85% compared with control. Surprisingly, there was no difference in fecal 3H-cholesterol excretion between the groups. Kinetic studies revealed increased catabolism/hepatic uptake of 3HDL-cholesteryl ether, resulting in no change in fecal HDL-cholesteryl ester excretion in the mice. To explore underlying mechanisms for the preservation of RCT despite low HDL levels in the EL-expressing mice, we investigated the effects of hepatic SR-BI (scavenger receptor class B type I) knockdown. RCT assay revealed that knockdown of SR-BI alone reduced fecal excretion of macrophage-derived 3H-cholesterol. Interestingly, hepatic EL overexpression under SR-BI inhibition further attenuated fecal tracer counts as compared with control. Finally, we observed that EL overexpression enhanced in vivo RCT under pharmacological inhibition of hepatic ABCA1 (ATP-binding cassette transporter A1) by probucol. Hepatic EL expression compensates for reduced macrophage-derived cholesterol efflux to plasma because of low HDL levels by promoting cholesterol excretion to bile/feces via an SR-BI pathway, maintaining overall RCT in vivo. In contrast, EL-modified HDL might negatively regulate RCT via hepatic ABCA1. Despite extreme hypoalphalipoproteinemia, RCT is maintained in EL-expressing mice via SR-BI/ABCA1-dependent pathways.

Thermogenic adipocytes promote HDL turnover and reverse cholesterol transport

Brown and beige adipocytes combust nutrients for thermogenesis and through their metabolic activity decrease pro-atherogenic remnant lipoproteins in hyperlipidemic mice. However, whether the activation of thermogenic adipocytes affects the metabolism and anti-atherogenic properties of high-density lipoproteins (HDL) is unknown. Here, we report a reduction in atherosclerosis in response to pharmacological stimulation of thermogenesis linked to increased HDL levels in APOE*3-Leiden.CETP mice. Both cold-induced and pharmacological thermogenic activation enhances HDL remodelling, which is associated with specific lipidomic changes in mouse and human HDL. Furthermore, thermogenic stimulation promotes HDL-cholesterol clearance and increases macrophage-to-faeces reverse cholesterol transport in mice. Mechanistically, we show that intravascular lipolysis by adipocyte lipoprotein lipase and hepatic uptake of HDL by scavenger receptor B-I are the driving forces of HDL-cholesterol disposal in liver. Our findings corroborate the notion that high metabolic activity of thermogenic adipocytes confers atheroprotective properties via increased systemic cholesterol flux through the HDL compartment.

Measurement of Macrophage-Specific In Vivo Reverse Cholesterol Transport in Mice.

Reverse cholesterol transport (RCT) is one of the main processes that is thought to protect against cardiovascular disease. RCT constitutes the removal of cholesterol from peripheral sites, its transport through the plasma compartment for delivery to the liver for excretion. Here, we describe an in vivo RCT method that incorporates these steps, measuring movement of cholesterol from macrophages to the plasma, the liver, and finally to the feces in mice.

Erratum to: Measurement of Macrophage-Specific In Vivo Reverse Cholesterol Transport in Mice.

Erratum to: Measurement of Macrophage-Specific In Vivo Reverse Cholesterol Transport in Mice.

Probucol oxidized products, spiroquinone and diphenoquinone, promote reverse cholesterol transport in mice

Probucol oxidized products, spiroquinone and diphenoquinone, promote reverse cholesterol transport in mice

Probucol-Oxidized Products, Spiroquinone and Diphenoquinone, Promote Reverse Cholesterol Transport in Mice.

Oxidized products of probucol, spiroquinone and diphenoquinone, were shown to increase cell cholesterol release and plasma high-density lipoprotein (HDL) by inhibiting degradation of ATP-binding cassette transporter A1. We investigated whether these compounds enhance reverse cholesterol transport in mice. Spiroquinone and diphenoquinone increased ATP-binding cassette transporter A1 protein (2.8- and 2.6-fold, respectively, P<0.01) and apolipoprotein A-I-mediated cholesterol release (1.4- and 1.4-fold, P<0.01 and P<0.05, respectively) in RAW264.7 cells. However, diphenoquinone, but not spiroquinone, enhanced cholesterol efflux to HDL (+12%, P<0.05), whereas both increased ATP-binding cassette transporter G1 protein, by 1.8- and 1.6-fold, respectively. When given orally to mice, both compounds significantly increased plasma HDL-cholesterol, by 19% and 20%, respectively (P<0.05), accompanied by an increase in hepatic and macrophage ATP-binding cassette transporter A1 but not ATP-binding cassette transporter G1. We next evaluated in vivo reverse cholesterol transport by injecting RAW264.7 cells labeled with (3)H-cholesterol intraperitoneally into mice. Both spiroquinone and diphenoquinone increased fecal excretion of the macrophage-derived (3)H-tracer, by 25% and 28% (P<0.01 and P<0.05), respectively. spiroquinone/diphenoquinone did not affect fecal excretion of HDL-derived (3)H-cholesterol, implying that macrophage-to-plasma was the most important step in spiroquinone/diphenoquinone-mediated promotion of in vivo reverse cholesterol transport. Finally, spiroquinone significantly reduced aortic atherosclerosis in apolipoprotein E null mice when compared with the vehicle. Spiroquinone and diphenoquinone increase functional ATP-binding cassette transporter A1 in both the macrophages and the liver, elevate plasma HDL-cholesterol, and promote overall reverse cholesterol transport in vivo. These compounds are promising as therapeutic reagents against atherosclerosis.

Abstract 10941: Hepatic Overexpression of Endothelial Lipase Induces a Markedly Low HDL-Cholesterol but Maintains Macrophage Reverse Cholesterol Transport in Mice in an ABCA1-dependent Fashion

We previously showed that hepatic overexpression of endothelial lipase (EL) by adenoviral vectors markedly reduced plasma HDL levels but maintained macrophage reverse cholesterol transport (RCT) in an SR-BI-dependent fashion in mice. In the present study, we further investigated roles of ABCA1 using probucol, an ABCA1 inhibitor, under EL overexpression. In vivo RCT assay demonstrated that hepatic EL overexpression reduced macrophages-derived 3H-cholesterol in plasma, especially HDL fractions but maintained fecal 3H-cholesterol excretion as compared with the control under normal chow. Probucol diet reduced both 3H- and non-radiolabeled-cholesterol levels in HDL fraction, which were further exacerbated by EL overexpression. Interestingly, probucol diet combined with EL overexpression clearly promoted fecal excretion of macrophages-derived 3H-cholesterol, indicating that EL overexpression and ABCA1 inhibition synergistically enhanced macrophage RCT despite of extremely low HDL levels.

Cyclosporine A Impairs the Macrophage Reverse Cholesterol Transport in Mice by Reducing Sterol Fecal Excretion

Despite the efficacy in reducing acute rejection events in organ transplanted subjects, long term therapy with cyclosporine A is associated with increased atherosclerotic cardiovascular morbidity. We studied whether this drug affects the antiatherogenic process of the reverse cholesterol transport from macrophages in vivo. Cyclosporine A 50 mg/kg/d was administered to C57BL/6 mice by subcutaneous injection for 14 days. Macrophage reverse cholesterol transport was assessed by following [3H]-cholesterol mobilization from pre-labeled intraperitoneally injected macrophages, expressing or not apolipoprotein E, to plasma, liver and feces. The pharmacological treatment significantly reduced the amount of radioactive sterols in the feces, independently on the expression of apolipoprotein E in the macrophages injected into recipient mice and in absence of changes of plasma levels of high density lipoprotein-cholesterol. Gene expression analysis revealed that cyclosporine A inhibited the hepatic levels of cholesterol 7-alpha-hydroxylase, concomitantly with the increase in hepatic and intestinal expression of ATP Binding Cassette G5. However, the in vivo relevance of the last observation was challenged by the demonstration that mice treated or not with cyclosporine A showed the same levels of circulating beta-sitosterol. These results indicate that treatment of mice with cyclosporine A impaired the macrophage reverse cholesterol transport by reducing fecal sterol excretion, possibly through the inhibition of cholesterol 7-alpha-hydroxylase expression. The current observation may provide a potential mechanism for the high incidence of atherosclerotic coronary artery disease following the immunosuppressant therapy in organ transplanted recipients.

Liver-Specific Cholesteryl Ester Hydrolase Deficiency Attenuates Sterol Elimination in the Feces and Increases Atherosclerosis in Ldlr −/− Mice

Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.

Scavenger receptor BI and ABCG5/G8 differentially impact biliary sterol secretion and reverse cholesterol transport in mice

Biliary lipid secretion plays an important role in gallstone disease and reverse cholesterol transport (RCT). Using Sr-bI/Abcg5 double knockout mice (dko), the present study investigated the differential contribution of two of the most relevant transporters: adenosine triphosphate (ATP)-binding cassette subfamily G member 5 and 8 (ABCG5/G8) and scavenger receptor class B type I (SR-BI) to sterol metabolism and RCT. Plasma cholesterol levels increased in the following order, mainly due to differences in high density lipoprotein (HDL): Abcg5 ko < wild type < Sr-bI/Abcg5 dko < Sr-bI ko. Liver cholesterol content was elevated in Sr-bI ko only (P < 0.05). In Sr-bI/Abcg5 dko plasma plant sterols were highest, while hepatic plant sterols were lower compared with Abcg5 ko (P < 0.05). Under baseline conditions, biliary cholesterol secretion rates decreased in the following order: wild type > Sr-bI ko (-16%) > Abcg5 ko (-75%) > Sr-bI/Abcg5 dko (-94%), all at least P < 0.05, while biliary bile acid secretion did not differ between groups. However, under supraphysiological conditions, upon infusion with increasing amounts of the bile salt tauroursodeoxycholic acid, Abcg5 became fully rate-limiting for biliary cholesterol secretion. Additional in vivo macrophage-to-feces RCT studies demonstrated an almost 50% decrease in overall RCT in Sr-bI/Abcg5 dko compared with Abcg5 ko mice (P < 0.01). These data demonstrate that (1) SR-BI contributes to ABCG5/G8-independent biliary cholesterol secretion under basal conditions; (2) biliary cholesterol mass secretion under maximal bile salt-stimulated conditions is fully dependent on ABCG5/G8; and (3) Sr-bI contributes to macrophage-to-feces RCT independent of Abcg5/g8.

RHDL administration increases reverse cholesterol transport in mice, but is not additive on top of ezetimibe or cholestyramine treatment

ObjectivePromoting reverse cholesterol transport (RCT) is a major atheroprotective property of HDL. The present study explored the effect of stimulating the first step of RCT (cholesterol efflux from macrophages) alone or in combination with stimulating the last step of RCT (fecal sterol excretion). Methods and resultsReconstituted HDL (rHDL) was injected into wild-type mice either with or without administration of the cholesterol absorption inhibitor ezetimibe or the bile acid sequestrant cholestyramine. Single dose administration of rHDL (100 mg apoA-I/kg) resulted in an early (4 h) increase in plasma free cholesterol levels (p < 0.001), without affecting hepatic cholesterol levels or fecal mass sterol excretion. rHDL injection also increased [3H]cholesterol appearance in plasma at an early time-point (4 h) after intraperitoneal administration of [3H]cholesterol-labeled mouse macrophage foam cells and fecal radioactivity excretion indicating completed RCT was increased by 26% (p < 0.05). Ezetimibe treatment inhibited intestinal cholesterol absorption by 74% (p < 0.01), but also the bile acid sequestrant cholestyramine decreased cholesterol absorption significantly (24%, p < 0.01). Consequently, ezetimibe increased RCT 2.1-fold (p < 0.001) primarily within fecal neutral sterols, while cholestyramine increased RCT by 3.6-fold (p < 0.001), primarily within bile acids (p < 0.001), but also within neutral sterols (p < 0.001). However, no additive effects of both intestinal sterol uptake inhibitors were observed on top of rHDL administration. ConclusionThese data demonstrate that increasing the first step of RCT by rHDL administration results in transient cholesterol mobilization from macrophages to plasma. This effect is not further enhanced by stimulating the last step of RCT, fecal sterol excretion.

Abstract 56: Macrophage Reverse Cholesterol Transport in Mice Relies on The Lymphatic Vasculature

Reverse cholesterol transport (RCT) refers to mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used two models of disrupted lymphatic drainage from skin -one surgical and the other genetic- to quantitatively track RCT following injection of [3H]cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analogue (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aorta of donor atherosclerotic apoE-/- mice with [2H]6-labeled cholesterol and surgically transplanting these aortas into recipient apoE-/- mice that were treated with anti-VEGFR3 mAb to block lymphatic regrowth or with control mAb to allow such regrowth. [2H]-Cholesterol was retained in aortas of anti-VEGFR3 treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. Therefore, supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice

Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin--1 surgical and the other genetic--to quantitatively track RCT following injection of [3H]-cholesterol-loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [2H]6-labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [2H]-Cholesterol was retained in aortae of anti-VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Stimulation of Hepatic Apolipoprotein A-I Production by Novel Thieno-Triazolodiazepines: Roles of the Classical Benzodiazepine Receptor, PAF Receptor, and Bromodomain Binding.

Expression and secretion of apolipoprotein A-I (apoA-I) by cultured liver cells can be markedly stimulated by triazolodiazepines (TZDs). It has been shown previously that the thieno-TZD Ro 11-1464 increases plasma levels of apoA-I and in vivomacrophage reverse cholesterol transport in mice. However, these effects were only seen at high doses, at which the compound could act on central benzodiazepine (BZD) receptors or platelet activating factor (PAF) receptors, interfering with its potential utility. In this work, we describe 2 new thieno-TZDs MDCO-3770 and MDCO-3783, both derived from Ro 11-1464. These compounds display the same high efficacy on apoA-I production, metabolic stability, and lack of cytotoxicity in cultured hepatocytes as Ro 11-1464, but they do not bind to the central BZD receptor and PAF receptor. The quinazoline RVX-208 was less efficacious in stimulating apoA-I production and displayed signs of cytotoxicity. Certain TZDs stimulating apoA-I production are now known to be inhibitors of bromodomain (BRD) extra-terminal (BET) proteins BRDT, BRD2, BRD3, and BRD4, and this inhibition was inferred as a main molecular mechanism for their effect on apoA-I expression. We show here that the thieno-TZD (+)-JQ1, a potent BET inhibitor, strongly stimulated apoA-I production in Hep-G2 cells, but that its enantiomer (−)-JQ1, which has no BET inhibitor activity, also showed considerable effect on apoA-I production. MDCO-3770 and MDCO-3783 also inhibited BRD3 and BRD4 in vitro, with potency somewhat below that of (+)-JQ1. We conclude that the effect of thieno-TZDs on apoA-I expression is not due to inhibition of the BZD or PAF receptors and is not completely explained by transcriptional repression by BET proteins.

Benign Prostate Hyperplasia Meets Liver X Receptor

Viennois and co-workers (1) characterized Lxr knockout mice that, interestingly, develop hyperplasia of the ventral prostate. The Lxr-knockout mice have the potential to develop into a novel and very important mouse model system for benign prostate hyperplasia (BPH) with similar symptoms as in human such as hyperproliferative epithelium and hypertrophic stroma. Such an in vivo model system would be a very useful tool to analyze the development and progression of BPH and perhaps in future also as a test system for therapeutic treatment of BPH. BPH is an increasingly important health problem of man. There is a rising incidence of hyperplasia of the prostate during age. An estimated 50% of men have histological evidence of BPH at the age of 50–60 yr and 75% at the age of 80 yr, whereas in 40–50% of these men, BPH becomes clinically significant (2). The hyperplasia of the prostate may compress the urethral canal causing partial or complete constriction of the urethra. This hinders the normal flow of urine. Also the bladder may be unable to be emptied completely, causing retention of urine in the bladder and subsequently strangury associated with lower quality of life. Furthermore, incomplete voiding can lead to growth of bacteria in the bladder and an increased risk of urinary tract infection. The main two currently used medication directions of BPH address on one hand the relaxation of smooth muscles such as by -blockers and on the other hand the reduction of dihydrotestosterone production using 5 -reductase inhibitors. The latter is used to inhibit the activation of the androgen receptor (AR; NR3C4), known as a major factor promoting growth of the normal prostate and also BPH. Interestingly, herbal medications are often used for treatment of BPH. These include extracts from saw palmetto, pumpkin seed (Cucurbita pepo), pollen extracts from rye grass (Secale cereale), stinging nettle (Urtica dioica), and the African plum tree (Prunus africana; Pygeum africanum). For most of these plant extracts, the active compounds are not yet discovered. From the bark of P. africanum, specific compounds have been identified that exhibit AR-antagonistic function, indicating that the bark contains natural herbal AR antagonists (3). Testosterone promotes prostate cell proliferation; however, relatively low levels of serum testosterone are found in patients with BPH (4, 5). This indicates that also other causes of BPH must be considered. Notably, the consumption of certain fatty acids, particularly of animal origin, has been correlated with increased prostate problems. High cholesterol levels and diabetes are associated with prostate cancer development (6, 7). This may suggest that cholesterol signaling could be involved in development and progression of BPH. The liver X receptors (LXR) are important regulators of cholesterol, fatty acid, and glucose homeostasis. LXR are, as the AR, members of the nuclear receptor superfamily of ligand-controlled transcription factors. LXR bind as ligands endogenously oxidized forms of cholesterol, the oxysterols, that are present in animal fat. Two isoforms of LXR have been identified and are referred to as LXR (NR1H3) and LXR (NR1H2) classified into the subfamily 1, the thyroid hormone receptor-like members of the nuclear receptor superfamily. The activation of LXR has been shown to inhibit cardiovascular disease and reduce atherosclerosis in animal models. Liver LXR expression is crucial for whole-body cholesterol homeostasis and reverse cholesterol transport in mice. LXR agonists improve glucose tolerance in a murine model of diet-induced obesity and insulin resistance by regulating genes involved in glucose metabolism in liver and adipose tissue (8, 9).