Intestinal cell-specific vitamin D receptor (VDR)-mediated transcriptional regulation of CYP3A4 gene.

CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene. In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRalpha heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4. Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRalpha heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells. We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.

Synthesis of fluorescent C24-ceramide: evidence for acyl chain length dependent differences in penetration of exogenous NBD-ceramides into human skin.

Topical skin lipid supplementation may provide opportunities for controlling ceramide (Cer) deficiency in skin diseases such as atopic dermatitis or psoriasis. Here we describe the synthesis of a long-chain 7-nitrobenzo[c][1,2,5]oxadiazol-4-yl (NBD)-labeled Cer and its different penetration through human skin compared to widely used short-chain fluorescent Cer tools.

Atorvastatin Increases Endoglin, SMAD2, Phosphorylated SMAD2/3 and eNOS Expression in ApoE/LDLR Double Knockout Mice.

AIM: Endoglin is a homodimeric transmembrane glycoprotein that has been demonstrated to affect transforming growth factor beta (TGF-beta) signaling and endothelial nitric oxide synthase (eNOS) expression by affecting SMAD proteins in vitro. Thus, in this study we stepped forward to elucidate whether endoglin is co-expressed with SMAD2, phosphorylated SMAD2/3 proteins and eNOS in vivo in atherosclerotic lesions in ApoE/LDLR double knockout mice. In addition, we sought whether endoglin expression as well as the expression of SMAD2, phosphorylated SMAD2/3 and eNOS is affected by atorvastatin treatment. METHODS: Two-month-old female ApoE/LDLR double knockout mice were divided into two groups. The control group was fed with the western type diet whereas in the atorvastatin group, atorvastatin at dose 100 mg/kg per day was added to the same diet. Immunohistochemical and western blot analysis of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS expressions in aorta were performed. RESULTS: The biochemical analysis showed that administration of atorvastatin significantly decreased level of total cholesterol, VLDL, LDL, TAG, and significantly increased level of HDL cholesterol. Fluorescence immunohistochemistry showed endoglin co-expression with SMAD2, phosphorylated SMAD2/3 and eNOS in aortic endothelium covering atherosclerotic lesions in both control and atorvastatin treated mice. Western blot analysis demonstrated that atorvastatin significantly increased expression of endoglin, SMAD2, phosphorylated SMAD2/3, and eNOS in mice aorta. CONCLUSION: These findings suggest, that endoglin might be interesting marker of endothelial dysfunction and/or atherogenesis which is upregulated by statins implicating potential beneficial role of endoglin and its pathway in atherosclerosis.

Expression and activity of vitamin D receptor in the human placenta and in choriocarcinoma BeWo and JEG-3 cell lines.

Vitamin D receptor (VDR) regulates the expression of many genes involved in mineral metabolism, cellular proliferation, differentiation and drug biotransformation. We studied the expression and activity of VDR and its heterodimerization partner retinoid X receptor-alpha (RXRalpha) in choriocarcinoma trophoblast cell lines BeWo and JEG-3, in comparison with human isolated placental cytotrophoblasts and human full term placenta. We found that VDR and RXRalpha are localised in the human term placenta trophoblast and expressed in isolated cytotrophoblasts. However, we found low expression and no transcriptional activity of VDR in used choriocarcinoma cell lines. The inhibitor of DNA methylation, 5-deoxy-3'-azacytidine, and histone deacetylase inhibitor sodium butyrate partially restored the expression of VDR, suggesting an epigenetic suppression of the gene in choriocarcinoma cells. Differentiation of BeWo cells resulted in up-regulation of VDR mRNA. Finally, we observed a non-genomic effect of 1,25(OH)(2)D(3) in the activation of the extracellular signal-regulated kinase (ERK) signalling pathway in JEG-3 cells. In conclusion, our results suggest an epigenetic repression of VDR gene expression and activity in choriocarcinoma cell lines, and a non-genomic effect of 1,25(OH)(2)D(3) in JEG-3 cells.

Microdispersed Oxidized Cellulose as a novel potential substance with hypolipidemic properties.

OBJECTIVE: The aim of this study was to determine whether Microdispersed Oxidized Cellulose (MDOC) possesses a hypolipidemic effect in apolipoprotein-E/low-density lipoprotein receptor double-knockout (ApoE/LDLR-deficient) mice and the possible mechanism of this effect in mice. METHODS: Female ApoE/LDLR-deficient mice subdivided into two groups were fed with a Western-type diet for 8 wk, and the experimental group was supplemented with 5% MDOC for 8 wk. Female C57BL/6J mice were fed an atherogenic diet containing 5% MDOC or pectin for the determination of a possible hypolipidemic mechanism of MDOC action. RESULTS: Biochemical analysis showed that 5% MDOC treatment significantly decreased total cholesterol by 20% (P = 0.0338) and very-LDL cholesterol by 21% (P = 0.0110) and significantly increased the level of high-density lipoprotein cholesterol by 62% (P = 0.0172) when compared with non-treated ApoE/LDLR-deficient mice. The results Association of Official Analytical Chemists method 991.43 revealed that MDOC contains 59.78 +/- 5.0% of fiber. Furthermore, it was demonstrated that administration of MDOC did not affect cholesterol absorption in the small intestine. Using C57BL/6J mice, MDOC and pectin treatments decreased cholesterol content in liver and increased fermentation in the gut in vivo. In vitro experiments confirmed that MDOC is fermentable under conditions mimicking those in the large intestine. CONCLUSION: We demonstrated hypolipidemic effects of MDOC in ApoE/LDLR-deficient mice. Moreover, we propose that MDOC is a hypolipidemic soluble fiber acting probably by increased fermentation and production of short-chain fatty acids in the large intestine in mice. We propose that MDOC might be a possible source of soluble fiber for use in dietary supplements.

Atorvastatin has hypolipidemic and anti-inflammatory effects in apoE/LDL receptor-double-knockout mice.

Statins are first-line pharmacotherapeutic agents for hypercholesterolemia treatment in humans. However the effects of statins in animal models of atherosclerosis are not very consistent. Thus we wanted to evaluate whether atorvastatin possesses hypolipidemic and anti-inflammatory effects in mice lacking apolipoprotein E/low-density lipoprotein receptor (apoE/LDLR-deficient mice). Two-month-old female apoE/LDLR-deficient mice (n=24) were randomly subdivided into 3 groups. The control group of animals (n=8) was fed with the western type diet (atherogenic diet) and in other two groups atorvastatin was added to the atherogenic diet at the dosage of either 10 mg/kg or 100 mg/kg per day for a period of 2 months. Biochemical analysis of lipids, ELISA analysis of monocyte chemotactic protein-1 (MCP-1) in blood, quantification of lesion size and expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) in the atherosclerotic lesion by means of immunohistochemistry and Western blot analysis were performed. The biochemical analysis showed that administration of atorvastatin (100 mg/kg/day) significantly decreased level of total cholesterol, lipoproteins (VLDL and LDL), triacylglycerol, and moreover significantly increased level of HDL. ELISA analysis showed that atorvastatin significantly decreased levels of MCP-1 in blood and immunohistochemical and Western blot analysis showed significant reduction of VCAM-1 and ICAM-1 expression in the vessel wall after atorvastatin treatment (100 mg/kg/day). In conclusion, we demonstrated here for the first time strong hypolipidemic and anti-inflammatory effects of atorvastatin in apoE/LDLR-deficient mice. Thus, we propose that apoE/LDLR-deficient mice might be a good animal model for the study of statin effects on potential novel markers involved in atherogenesis and for the testing of potential combination treatment of new hypolipidemic substances with statins.

Endothelial expression of endoglin in normocholesterolemic and hypercholesterolemic C57BL/6J mice before and after atorvastatin treatment.

Endoglin (CD105) is a homodimeric transmembrane glycoprotein strongly related to transforming growth factor (TGF)-beta signaling and many pathological states. In this study, we wanted to evaluate whether endoglin is expressed in normocholesterolemic and hypercholesterolemic C57BL/6J mice as well as whether it is affected by atorvastatin treatment in these mice. C57BL/6J mice were fed with chow diet or an atherogenic diet for 12 weeks after weaning. In 2 atorvastatin-treated groups, mice were fed the same diets (chow or atherogenic) as described above except atorvastatin was added at the dosage of 10 mg x kg(-1) x day(-1) for the last 8 weeks before euthanasia. Biochemical analysis of blood samples revealed that administration of atherogenic diet significantly increased levels of total cholesterol, VLDL, LDL, and decreased levels of HDL. Atorvastatin treatment resulted in a significant decrease in total cholesterol and VLDL only in mice fed by atherogenic diet. Quantitative stereological analysis revealed that atorvastatin significantly decreased endothelial expression of endoglin in C57BL/6J mice fed the atherogenic diet. In conclusion, we demonstrated that endothelial expression of endoglin is upregulated by hypercholesterolemia and decreased by the hypolipidemic effect of atorvastatin in C57BL/6J mice, suggesting that endoglin expression could be involved in atherogenesis.

Atorvastatin has distinct effects on endothelial markers in different mouse models of atherosclerosis.

PURPOSE: Atherosclerosis is a progressive process that initially involves endothelial dysfunction. We investigated the effects of atorvastatin on both lipid parameters, and VCAM-1 and ICAM-1 expression in apoE-deficient or wild type C57BL/6J mice. METHODS: The C57BL/6J mice were fed with either chow or an atherogenic diet for 12 weeks. Male apoE-deficient mice were fed with the chow diet for 12 weeks. In 3 atorvastatin treated groups mice were fed the same diet as described above except atorvastatin was added to the diet at the dosage of 10 mg/kg per day for the last 8 weeks before euthanasia. RESULTS: Biochemical analysis showed that atorvastatin significantly decreased total cholesterol levels and VLDL in C57BL/6J mice fed with atherogenic diet but increased serum lipid levels in apoE-deficient mice. Stereological analysis of the immunohistochemical staining revealed that atorvastatin reduced endothelial expression of ICAM-1 and VCAM-1 only in C57BL/6J mice on chow diet. CONCLUSION: We have demonstrated that endothelial expression of both VCAM-1 and ICAM-1 does not correlate with cholesterol levels in these mice. Moreover, we showed that 8-week administration of atorvastatin decrease endothelial expression of VCAM-1 and ICAM-1 in C57BL/6J wild type mice beyond its lipid lowering effect but not in C57BL/6J wild type mice fed by atherogenic diet or in apoE-deficient mice.

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line.

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.

MDOC and atorvastatin have potential antiinflammatory effects in vascular endothelium of apoE-/- mouse model of atherosclerosis.

Members of the immunoglobulin superfamily of endothelial adhesion molecules, vascular cell adhesion molecule (VCAM-1) and intercellular cell adhesion molecule (ICAM- 1), strongly participate in leukocyte adhesion to the endothelium and play an important role in all stages of atherogenesis. The aim of this study was to detect and quantify the changes of endothelial expression of VCAM-1, and ICAM-1 in the vessel wall after the short-term administration of simvastatin, atorvastatin, and micro dispersed derivatives of oxidised cellulose (MDOC) in apolipoprotein-E-deficient (apoE(-/-)) mice atherosclerotic model. Hyperlipidemic apoE(-/-) mice (n = 32) received normal chow diet or diet containing simvastatin or atorvastatin 10 mg/kg/day or MDOC 50 mg/kg/day. Total cholesterol, VLDL, LDL, HDL and TAG were measured and the endothelial expression of VCAM-1 and ICAM-1 was visualized and quantified by means of immunohistochemistry and stereology, respectively. Total cholesterol levels was insignificantly lowered only in MDOC treated mice but not in mice treated with statins. ICAM-1 endothelial expression was not affected by neither simvastatin nor MDOC treatment. However, significant diminution of VCAM-1 endothelial expression was observed in both atorvastatin and MDOC treated mice. These results provide new information of potential hypolipidemic substance MDOC and its potential anti-inflammatory effects. Furthermore, we have confirmed anti-inflammatory effects of atorvastatin independent of plasma cholesterol lowering. Thus, the results of this study show potential benefit of both MDOC and atorvastatin treatment in apoE(-/-) mouse model of atherosclerosis suggesting their possible combination might be of interest.

Endoglin expression in hyper-cholesterolemia and after atorvastatin treatment in apoE-deficient mice.

PURPOSE: Endoglin (CD105) is a marker of activated endothelium and a modulator of TGF-beta signaling. We hypothesized whether endothelial expression of endoglin is changed in hypercholesterolemia as well as whether its expression is affected by atorvastatin treatment in apoE-deficient mice. METHODS: ApoE-deficient mice were fed with the chow diet for either 4 weeks or for 12 weeks respectively. In two treated groups, mice were fed with chow diet except atorvastatin was added to the diet for the last 4 weeks or for the last 8 weeks respectively, before euthanasia. RESULTS: Administration of atorvastatin did not affect lipid parameters after 4 weeks treatment, however increased all lipid parameters after 8 weeks of treatment. Stereological analysis of immunohistochemical staining revealed that atorvastatin significantly decreased endoglin expression in endothelium after 4 weeks of treatment but increased it after 8 weeks of treatment. CONCLUSIONS: This study demonstrates that endoglin is expressed by aortic endothelium showing similar staining patterns like other markers involved in the process of atherosclerosis. In addition, we showed that endoglin expression in endothelium could be affected by the administration of atorvastatin beyond its lipid lowering effects in apoE-deficient mice.