Oxysterol Production Research Articles

A combined transcriptomics and proteomics approach reveals S100A4 as a potential biomarker for Graves' orbitopathy.

There are no reliable biomarkers to identify Graves' disease patients who will develop severe Graves' orbitopathy (GO). We hypothesize that integrating various omics platforms can enhance our understanding of disease mechanisms and uncover potential biomarkers. This study aimed to (1) elucidate the differential gene expression profile of orbital fibroblasts in GO during early adipogenesis to better understand disease mechanisms and (2) compare tear protein profiles from our earlier study and the transcriptome profiles of orbital fibroblasts (OFs) to identify possible biomarkers of the disease. OFs were grown from orbital adipose tissue obtained from nine GO patients (three for discovery and six for validation experiments). Total RNA was extracted from OFs on day 0 as the baseline for each sample and from differentiated OFs on days 4 and 8. Protein-protein interaction (PPI) analysis and functional enrichment analysis were also carried out. The differentially expressed genes (DEGs) from the RNA sequencing experiments were then compared to the full tear proteome profile from the author's previous study, which examined the tear protein changes of GO patients based on fold change > 1.6 or < -1.6. FDR < 0.05 was applied within all datasets. Further validation of S100 calcium-binding protein A4 (S100A4) downregulation in GO was performed via quantitative real-time PCR (qPCR). The whole transcriptomic analysis revealed 9 upregulated genes and 15 downregulated genes in common between the discovery and validation experiments. From the PPI network analysis, an interaction network containing six identified DEGs (ALDH2, MAP2K6, MT2A, SOCS3, S100A4, and THBD) was observed. The functional enrichment network analysis identified a set of genes related to oxysterol production. S100A4 was found to be consistently downregulated in both our transcriptome studies and the full-tear proteome profile from the author's previous study. Our study identified several DEGs and potential gene pathways in GO patients, which concurred with the results of other studies. Tear S100A4 may serve as a biomarker for the propensity to develop thyroid eye disease (TED) in patients with autoimmune thyroid disease (AITD) before clinical manifestation and should be confirmed in future studies.

Chemical synthesis and biochemical properties of cholestane-5α,6β-diol-3-sulfonate: A non-hydrolysable analogue of cholestane-5α,6β-diol-3β-sulfate

Cholestane-3β,5α,6β-triol (CT) is a primary metabolite of 5,6-epoxycholesterols (5,6-EC) that is catalyzed by the cholesterol-5,6-epoxide hydrolase (ChEH). CT is a well-known biomarker for Niemann-Pick disease type C (NP-C), a progressive inherited neurodegenerative disease. On the other hand, CT is known to be metabolized by the 11β-hydroxysteroid-dehydrogenase of type 2 (11β-HSD2) into a tumor promoter named oncosterone that stimulates the growth of breast cancer tumors. Sulfation is a major metabolic transformation leading to the production of sulfated oxysterols. The production of cholestane-5α,6β-diol-3β-O-sulfate (CDS) has been reported in breast cancer cells. However, no data related to CDS biological properties have been reported so far. These studies have been hampered because sulfate esters of sterols and steroids are rapidly hydrolyzed by steroid sulfatase to give free steroids and sterols. In order to get insight into the biological properties of CDS, we report herein the synthesis and the characterization of cholestane-5α,6β-diol-3β-sulfonate (CDSN), a non-hydrolysable analogue of CDS. We show that CDSN is a potent inhibitor of 11β-HSD2 that blocks oncosterone production on cell lysate. The inhibition of oncosterone biosynthesis of a whole cell assay was observed but results from the blockage by CDSN of the uptake of CT in MCF-7 cells. While CDSN inhibits MCF-7 cell proliferation, we found that it potentiates the cytotoxic activity of post-lanosterol cholesterol biosynthesis inhibitors such as tamoxifen and PBPE. This effect was associated with an increase of free sterols accumulation and the appearance of giant multilamellar bodies, a structural feature reminiscent of Type C Niemann-Pick disease cells and consistent with a possible inhibition by CDSN of NPC1. Altogether, our data showed that CDSN is biologically active and that it is a valuable tool to study the biological properties of CDS and more specifically its impact on immunity and viral infection.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

Commentary on 'Identification and characterization of select oxysterols as ligands for GPR17'.

An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells.

Immunoglobulin A (IgA) secretion by plasma cells, terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. Exposure to diet-derived and commensal-derived signals provides immune cells with organizing cues that instruct their effector function and dynamically shape intestinal immune responses at the mucosal barrier. Recent data have described metabolic and microbial inputs controlling T cell and innate lymphoid cell activation in the gut; however, whether IgA-secreting lamina propria plasma cells are tuned by local stimuli is completely unknown. Although antibody secretion is considered to be imprinted during B cell differentiation and therefore largely unaffected by environmental changes, a rapid modulation of IgA levels in response to intestinal fluctuations might be beneficial to the host. In the present study, we showed that dietary cholesterol absorption and commensal recognition by duodenal intestinal epithelial cells lead to the production of oxysterols, evolutionarily conserved lipids with immunomodulatory functions. Using conditional cholesterol 25-hydroxylase deleter mouse line we demonstrated that 7α,25-dihydroxycholesterol from epithelial cells is critical to restrain IgA secretion against commensal- and pathogen-derived antigens in the gut. Intestinal plasma cells sense oxysterols via the chemoattractant receptor GPR183 and couple their tissue positioning with IgA secretion. Our findings revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around plasma cell localization for efficient mucosal protection.

Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury.

Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level. Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols, which have profound effects on neuropathology. 25-Hydroxycholesterol (25-HC), the main product of the membrane-associated enzyme cholesterol-25-hydroxylase (CH25H), plays important roles in mediating neuroinflammation. However, whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC, as well as the resulting pathological effects, remain unclear. In the present study, spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression. A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo. In cultured primary astrocytes, thrombin interacted with protease-activated receptor 1, mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway. Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration. Finally, injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function. Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.

Epithelial-derived oxysterol production tunes intestinal IgA secretion against commensals and enteric pathogen in tissue

Abstract Immunoglobulin A (IgA) secretion by plasma cells (PCs), terminally differentiated B cells residing in the intestinal lamina propria, assures microbiome homeostasis and protects the host against enteric infections. However, whether exposure to diet-derived and commensal-derived signals instruct tissue resident PCs effector function and dynamically shape IgA immune responses at the mucosal barrier remain largely uninvestigated. Here, we demonstrated that intestinal epithelial cells (IECs) integrate luminal input to produce 7α,25-dihydroxycholesterol (7α,25-HC), a cholesterol metabolite (oxysterol) with immunomodulatory functions. In IECs, both cholesterol uptake via NPC1L1 and commensal recognition via MyD88 controlled oxysterol production. Mice lacking the oxysterol enzyme CH25H specifically in IECs abolished 7α,25-HC production and allowed to study oxysterol generation and activity in the small intestine. Inability of IECs to generate 7α,25-HC enhanced IgA secretion by PCs in the gut, suggesting that oxysterol negatively regulate humoral response at the mucosal barriers. Mechanistically, we showed that intestinal PCs sensed 7α,25-HC via the chemoattractant receptor GPR183 to position in the lamina propria tissue. This IEC-PC axis was rapidly modulated by cholesterol dietary content and tuned Salmonella-specific IgA response. Our finding revealed a new mechanism linking dietary cholesterol and humoral immune responses centered around PC localization for efficient mucosal protection. Supported by grants from Kenneth Rainin Foundation, Innovator Award, Charles H. Hood Foundation Child Health Research Awards Program, The Leukemia and Lymphoma Society New Idea Award and the Multiple Myeloma Research Fellowship, NIH ( AI40098 ) and The American Association of Immunologists Careers in Immunology Fellowship Program.

Transcription of cytochrome P450 46A1 in NIH3T3 cells is negatively regulated by FBS

Extracellular administration of side-chain oxysterols, such as 24S-hydroxycholesterol (24S-HC), 27-hydroxycholesterol (27-HC) and 25-hydroxycholesterol (25-HC) to cells suppresses HMG-CoA reductase (Hmgcr) and CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) mRNA levels. Oxysterols are enzymatically produced in cells from cholesterol by cytochrome P450 46A1 (Cyp46A1), Cyp27A1, Cyp3A11 and cholesterol 25-hydroxylase (Ch25h). We analyzed which of these oxysterol-producing enzymes are expressed in NIH3T3 cells and found that only Cyp46A1 was expressed. When Cyp46A1 was overexpressed in NIH3T3 cells, intrinsic oxysterols increased in the order 24S-HC > 25-HC > 27-HC. We investigated the mechanism regulating the production of endogenous oxysterols in NIH3T3 cells by Cyp46A1 and found that the mRNA, relative protein levels and enzymatic activity of Cyp46A1, and the amounts of 24S-HC, 25-HC and 27-HC significantly increased under serum-starved conditions, and these increases were suppressed by FBS supplementation. The aqueous phase of FBS obtained by the Bligh & Dyer method significantly suppressed Cyp46A1 mRNA levels. Fractionation of the aqueous phase by HPLC and analysis of the inhibiting fractions by nanoLC and TripleTOF MS/MS identified insulin-like factor-II (IGF-II). Cyp46A1 mRNA levels in serum-starved NIH3T3 cells were significantly suppressed by the addition of IGFs and insulin and endogenous oxysterol levels were decreased. CYP46A1 mRNA levels in the T98G human glioblastoma cell line were also increased by serum starvation but not by FBS supplementation, and the aqueous phase did not inhibit the increase. These results suggest that mRNA levels of Cyp46A1 are regulated by factors in FBS.

Cancer-derived cholesterol sulfate is a key mediator to prevent tumor infiltration by effector T cells.

Effective tumor immunotherapy requires physical contact of T cells with cancer cells. However, tumors often constitute a specialized microenvironment that excludes T cells from the vicinity of cancer cells, and its underlying mechanisms are still poorly understood. DOCK2 is a Rac activator critical for migration and activation of lymphocytes. We herein show that cancer-derived cholesterol sulfate (CS), a lipid product of the sulfotransferase SULT2B1b, acts as a DOCK2 inhibitor and prevents tumor infiltration by effector T cells. Using clinical samples, we found that CS was abundantly produced in certain types of human cancers such as colon cancers. Functionally, CS-producing cancer cells exhibited resistance to cancer-specific T-cell transfer and immune checkpoint blockade. Although SULT2B1b is known to sulfate oxysterols and inactivate their tumor-promoting activity, the expression levels of cholesterol hydroxylases, which mediate oxysterol production, are low in SULT2B1b-expressing cancers. Therefore, SULT2B1b inhibition could be a therapeutic strategy to disrupt tumor immune evasion in oxysterol-non-producing cancers. Thus, our findings define a previously unknown mechanism for tumor immune evasion and provide a novel insight into the development of effective immunotherapies.

Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

Anti-Inflammatory and Proliferative Properties of Luteolin-7-O-Glucoside.

Flavonoids display a broad range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages. Recent data showed their activity, and in particular of luteolin-7-O-glucoside (LUT-7G), in reduction of oxidative stress and inflammatory mechanisms in different physiological systems. In this paper, we tried to elucidate how LUT-7G could exert both antioxidant and anti-inflammatory effects in endothelial cells cultured in vitro. Here, we showed that LUT-7G is able to inhibit the STAT3 pathway, to have an antiproliferative action, and an important antioxidant property in HUVEC cells. These properties are exerted by the flavone in endothelial through the transcriptional repression of a number of inflammatory cytokines and their receptors, and by the inhibition of ROS generation. ROS and STAT3 activation has been correlated with the production of oxysterols and other hydroxylated fatty acids, and they have been recognized important as players of atherogenesis and cardiocirculatory system diseases. The analysis of the general production pathway of these hydroxylated species, showed a strong decrease of cholesterol hydroxylated species such as 7-alpha-hydroxicholesterol, 7-beta-hydroxicholesterol by the treatment with LUT-7G. This confirms the anti-inflammatory properties of LUT-7G also in the endothelial district, showing for the first time the molecular pathway that verify previous postulated cardiovascular benefits of this flavone.

Cholesterol-modifying drugs in COVID-19.

Infection with severe acute respiratory syndrom coronavirus 2 (SARS-CoV-2) is more likely to lead to poor outcomes in the elderly and those with cardiovascular disease, obesity or metabolic syndrome. Here, we consider mechanisms by which dyslipidaemia and the use of cholesterol-modifying drugs could influence the virus–host relationship. Cholesterol is essential for the assembly, replication and infectivity of enveloped virus particles; we highlight several cholesterol-modifying drugs with the potential to alter the SARS-CoV-2 life cycle that could be tested in in vitro and in vivo models. Although cholesterol is an essential component of immune cell membranes, excess levels can dysregulate protective immunity and promote exaggerated pulmonary and systemic inflammatory responses. Statins block the production of multiple sterols, oxysterols and isoprenoids, resulting in a pleiotropic range of context-dependent effects on virus infectivity, immunity and inflammation. We highlight antiviral, immunomodulatory and anti-inflammatory effects of cholesterol-modifying drugs that merit further consideration in the management of SARS-CoV-2 infection.

Is 24(S)-hydroxycholesterol a potent modulator of cholesterol metabolism in Müller cells? An in vitro study about neuron to glia communication in the retina

Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Müller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Müller cells were treated with 0, 0.5 or 1.5 μM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Müller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRβ). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Müller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Müller cells, leading to cholesterol depletion (-37 % at 1.5 μM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 μM) and lathosterol (-84 % at 1.5 μM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5μM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 μM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 μM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Müller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Müller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.

Oxysterol concentrations are associated with cholesterol concentrations and anemia in pediatric patients with sickle cell disease

Sickle cell disease (SCD) causes anemia, oxidative stress, chronic inflammation, and lipid abnormalities. Oxysterols are oxidized derivatives of cholesterol and affect cholesterol metabolism and eryptosis. Our aim was to determine whether the plasma concentrations of 7-ketocholesterol (7-KC) and cholestane-3β,5α,6β-triol (C-triol) were associated with hemolysis and lipid profile in patients with SCD. A total of 32 steady-state pediatric patients with SCD (22 HbSS and 10 HbSß+) and 25 healthy controls were included in the study. Hemolysis parameters, ferritin, serum iron, lipids, 7-KC and C-triol concentrations of all subjects were measured. Oxysterols were quantified with N,N-dimethylglycine derivatization via LC-MS/MS. 7-KC and C-triol concentrations were found to be increased in SCD patients, while there was no difference between the HbSS and HbSß+ subgroups. 7-KC concentrations s were correlated negatively with hemoglobin and positively with lactate dehydrogenase concentrations, while C-triol concentrations were negatively correlated with HDL cholesterol. Furthermore, while 7-KC and C-triol concentrations were highly correlated among controls, there was no correlation in patients. The findings of our study suggest that 7-KC and C-triol may have a role in SCD pathophysiology. The lack of correlation in patients’ 7-KC and C-triol concentrations suggest alterations in oxysterol production in patients with SCD.

2125-P: Increased StAR (Steroidogenic Acute Regulatory Protein) Is Detrimental to ß Cells and Promotes Mitochondrial Dysfunction

StAR is a cholesterol transport protein that transports cholesterol from the cytoplasm into the mitochondrial matrix for further metabolism by CYP enzymes. Depending on the CYP expressed, this results in production of corticosteroids, gonadal steroids, neurosteroids or oxysterols. These products generally exert positive effects on cellular health and function but can also be harmful. We previously showed that StAR is produced in β cells and is increased under conditions of amyloid formation in human islet amyloid polypeptide (hIAPP) transgenic mouse islets, a model of human islet amyloidosis in type 2 diabetes (T2D). Further, CYP11A1, CYP11B1, CYP7A1 and CYP17A1 are not expressed in mouse islets, and CYP27A1 is expressed but decreases with amyloid deposition. Therefore, we hypothesized that increased StAR in islets is deleterious to β-cell health as the downstream CYP enzymes are not present to metabolize mitochondrial cholesterol. We first demonstrated (by immunoblot) that StAR is expressed in human islets, a requirement if it is to play a role in T2D. Also in human islets, only CYP27A1 is detectable, while CYP11A1 and CYP7A1 are not (n=4). We next used a lentivirus to increase StAR expression (or a control lentivirus) in INS-1 cells, which resulted in reduced mitochondrial membrane potential (100±2.0% vs. 78.1±1.2%, n=2, p&amp;lt;0.05) and cell viability (100±4.1% vs. 89.7±7.7%, n=3, p&amp;lt;0.05). Finally, we determined whether reducing StAR expression with an siRNA oligonucleotide in hIAPP transgenic islets had beneficial effects. A 40% knockdown in StAR expression did not alter amyloid deposition, but tended to increase islet viability (100±3.1% vs. 134.7±12.2%, n=2, p=0.1). In summary, StAR is expressed in human islets and increasing its expression is deleterious due to changes in mitochondrial function and β-cell survival. Thus, reducing StAR expression and/or increasing expression of downstream CYP enzymes may be a novel strategy to protect β cells in T2D. Disclosure M.F. Hogan: None. N. Esser: None. A.T. Templin: None. S. Zraika: Research Support; Self; Novartis Pharmaceuticals Corporation. R.L. Hull: Research Support; Self; Boehringer Ingelheim Pharmaceuticals, Inc. S. El-Osta: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Intarcia Therapeutics, Inc., Janssen Pharmaceuticals, Inc., Merck &amp; Co., Inc., Novo Nordisk A/S. Consultant; Self; Neurimmune. Other Relationship; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Merck &amp; Co., Inc., Novo Nordisk A/S. Funding American Diabetes Association (1-18-PDF-174 to M.F.H.); National Institutes of Health (T32HL007028, F32DK109584); U.S. Department of Veterans Affairs (BX001060); University of Washington

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

The isoflavones genistein and daidzein increase hepatic concentration of thyroid hormones and affect cholesterol metabolism in middle-aged male rats

We examined whether isoflavones interfere with thyroid homeostasis, increase hepatic thyroid hormone concentrations and affect cholesterol metabolism in middle-aged (MA) male rats. Thirteen-month-old Wistar rats were injected subcutaneously with 35 mg/kg b.w./day of genistein, daidzein or vehicle (controls) for four weeks. Hepatic Dio1 gene expression was up-regulated by 70% (p < 0.001 for both) and Dio1 enzyme activity increased by 64% after genistein (p < 0.001) and 73% after daidzein treatment (p < 0.0001). Hepatic T3 was 75% higher (p < 0.05 for both), while T4 increased only after genistein treatment. Serum T4 concentrations were 31% lower in genistein- and 49% lower in dadzein-treated rats (p < 0.001 for both) compared with controls. Hepatic Cyp7a1 gene expression was up-regulated by 40% after genistein and 32% after daidzein treatment (p < 0.05 for both), in agreement with a 7α-hydroxycholesterol increase of 50% (p < 0.01) and 88% (p < 0.001), respectively. Serum 24- and 27-hydroxycholesterol were 30% lower (p < 0.05 for both), while only 24-hydroxycholesterol was decreased in the liver by 45% after genistein (p < 0.05) and 39% (p < 0.01) after dadzein treatment. Serum concentration of the cholesterol precursor desmosterol was 32% (p < 0.05) lower only after dadzein treatment alone, while both isoflavones elevated this parameter in the liver by 45% (p < 0.01). In conclusion, isoflavones increased T3 availability in the liver of MA males, despite decreasing serum T4. Hepatic increase of T3 possibly contributes to activation of the neutral pathway of cholesterol degradation into bile acids in the liver. While isoflavones obviously have the potential to trigger multiple mechanisms involved in cholesterol metabolism and oxysterol production, they failed to induce any hypocholesterolemic effect.

Heme Oxygenase-1 and Brain Oxysterols Metabolism Are Linked to Egr-1 Expression in Aged Mice Cortex, but Not in Hippocampus.

Throughout life, stress stimuli act upon the brain leading to morphological and functional changes in advanced age, when it is likely to develop neurodegenerative disorders. There is an increasing need to unveil the molecular mechanisms underlying aging, in a world where populations are getting older. Egr-1 (early growth response 1), a transcriptional factor involved in cell survival, proliferation and differentiation – with a role also in memory, cognition and synaptic plasticity, can be implicated in the molecular mechanism of the aging process. Moreover, Heme Oxygenase-1a (HO), a 32 kDa heat-shock protein that converts heme to iron, carbon monoxide and biliverdin, is a key enzyme with neuroprotective properties. Several in vitro and in vivo studies reported that HO-1 could regulate the metabolism of oxysterols, oxidation products of cholesterol that include markers of oxidative stress. Recently, a link between Egr-1 and HO-1 has been demonstrated in mouse lung cells exposed to cigarette smoke. In view of these data, we wanted to investigate whether Egr-1 can be implicated also in the oxysterol metabolism during brain aging. Our results show that Egr-1 expression is differently expressed in the cortex and hippocampus of old mice, as well as the oxysterol profile between these two brain areas. In particular, we show that the cortex experiences in an age-dependent fashion increasing levels of the Egr-1 protein, and that these correlate with the level of HO-1 expression and oxysterol abundance. Such a situation was not observed in the hippocampus. These results are further strenghtened by our observations made with Egr-1 KO mice, confirming our hypothesis concerning the influence of Egr-1 on oxysterol production and accumulation via regulation of the expression of HO-1 in the cortex, but not the hippocampus, of old mice. It is important to notice that most of the oxysterols involved in this process are those usually stimulated by oxidative stress, which would then represent the triggering factor for this mechanism.

Cilia-Associated Oxysterols Activate Smoothened

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.

79. LXR and ABC-transporter-expression patterns in the placenta in IUGR

Introduction Atherogenic lipid oxidation and cholesterol accumulation is increased in the placenta in IUGR. Both, hypoxic conditions and the formation of small molecules like oxysterols however are involved in cellular cholesterol homeostasis by regulating the cholesterol efflux ABC-transporter expression ABCA1 and ABCG1 via activating liver-X-receptors (LXR). Objectives/hypothesis Since it is assumed that hypoxia and oxysterol production or accumulation is increased in the IUGR placenta we questioned whether the LXR-ABCA1 pathway is activated to counteract cholesterol excess. Methodes Placentas of 40 IUGR and 40 controls (CTRL) arranged in tissue micro arrays (TMA) were analyzed immunohistochemically for LXRa, LXRb, ABCA1, and ABCG1. The expression was assessed in trophoblasts and endothelium cells semi-quantitatively using Immunoreactivity-Score (IRS). Expression values were related to maternal and fetal lipid profiles. Expression patterns were statistically accomplished via the Mann–Whitney test. Results The LXRa und ABCA1-expression was increased in trophoblasts as compared to controls (LXRa Median IRS (95%CI) IUGR = 6.3 (5.59–7.06), CTRL = 4.8 (4.51–5.85), p = 0.0207; ABCA1 Median IRS (95%CI) IUGR = 7.0 (6.33–7.87), CTRL = 3.9 (3.87–5.67), p = 0.0001). No differences were found in LXRb and ABCG1 expression patterns. Discussion An increased expression of cholesterol transport molecules in trophoblasts may be a compensatory mechanism against toxic effects of cholesterol excess and oxidation within the placenta.

Associations of gender and serum total cholesterol with CD4+ T cell count and HIV RNA load in antiretroviral-na\xefve individuals in Addis Ababa

BackgroundMales are more susceptible than females to infections due to the differences in endocrine-immune interactions. Furthermore, it is reported that lowering cell cholesterol impairs viral replication and infection in vitro. However, the production of oxysterols in vivo by oxidation of cholesterol may result in inhibition of HIV replication. Therefore, this study was designed to determine the associations of gender and serum total cholesterol with CD4+ T cell counts and/or WHO clinical stages, and HIV ribonucleic acid (RNA) load in antiretroviral therapy (ART) naive study population with known sero-positive time of stay in Addis Ababa. MethodsA cross-sectional study was conducted from February to August 2013 on 594 HIV-1 infected ART-naïve adult study participants in four hospitals Addis Ababa. CD4+ T-cell count, HIV RNA load, hemoglobin and fasting serum total cholesterol were determined. Socio-demographic characteristics, WHO clinical stages, and height and weight were collected from patients’ chart and triangulated by structured questionnaire. Pearson chi-square test, Spearman rank correlation and univariate and multivariate linear/logistic regression analyses were carried out to determine associations.ResultsMean HIV RNA load was found to be lower in women than in men (p < 0.05). CD4+ T cell count and serum total cholesterol were found to be significantly correlated with HIV RNA load (p < 0.01). Women were at lower risk of having higher HIV RNA load in comparison to men. In addition, having lower concentrations of serum total cholesterol was found to be independent predictor of higher HIV RNA load in comparison to those with higher concentrations of cholesterol in serum (p < 0.05). The multivariate binomial logistic regression also showed that the immune status was better in women than men, and in the presence of higher serum total cholesterol (p < 0.05).ConclusionGender and serum total cholesterol were found to be associated and independent predictors of HIV RNA load, and CD4+ cell count and/or WHO clinical stages. There is a significant lower HIV RNA load and better CD4+ T cell count in women and those study participants with higher serum total cholesterol.