apoE mRNA Research Articles

Binding and Repressive Activities of Apolipoprotein E3 and E4 Isoforms on the Human ApoD Promoter

Apolipoprotein D (ApoD) gene expression is increased in several neurological disorders such as Alzheimer’s disease (AD) and multiple sclerosis. We previously showed that transgenic mice that overexpress human ApoD show a better resistance against paraquat or OC43 coronavirus-induced neurodegeneration. Here, we identified several nuclear factors from the cortex of control and OC43-infected mice which bind a fragment of the proximal ApoD promoter in vitro. Of interest, we detected apolipoprotein E (ApoE). Human ApoE consists of three isoforms (E2, E3, and E4) with the E4 and E2 alleles representing a greater and a lower risk for developping AD, respectively. Our results show that ApoE is located in the nucleus and on the ApoD promoter in human hepatic and glioblastoma cells lines. Furthermore, overexpression of ApoE3 and ApoE4 isoforms but not ApoE2 significantly inhibited the ApoD promoter activity in U87 cells (E3/E3 genotype) cultured under normal or different stress conditions while ApoE knock-down by siRNA had a converse effect. Consistent with these results, we also demonstrated by ChIP assay that E3 and E4 isoforms, but not E2, bind the ApoD promoter. Moreover, using the Allen Brain Atlas in situ hybridization database, we observed an inverse correlation between ApoD and ApoE mRNA expression during development and in several regions of the mouse brain, notably in the cortex, hippocampus, plexus choroid, and cerebellum. This negative correlation was also observed for cortex layers IV–VI based on a new Transcriptomic Atlas of the Mouse Neocortical Layers. These findings reveal a new function for ApoE by regulating ApoD gene expression.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-013-8456-0) contains supplementary material, which is available to authorized users.

Abstract 4: Apolipoprotein A-iv Enhances Triglyceride Secretion and Reduces Hepatic Lipid Content by Promoting Apob Containing-lipoprotein Particle Expansion in Livers of Steatotic Mice

Apolipoprotein A-IV (apoA-IV) is a lipid binding protein expressed in mammalian intestine, as well as rodent liver. While previous in vitro studies demonstrated that apoA-IV promotes cellular triglyceride (TG) secretion by modulating apoB secretory trafficking and lipoprotein size, evidence that apoA-IV affects TG secretion in vivo is lacking. To explore the possible role of apoA-IV in controlling hepatic VLDL-mediated lipid efflux we assessed whether apoA-IV is regulated by liver lipid accumulation and if its deficiency or overexpression affects hepatic TG secretion. Under conditions of high fat diet-induced hepatic steatosis and steatosis caused by transgenic overexpression of SREBP-1a (SREBP-1aTg), a ~40-fold induction of hepatic apoA-IV mRNA and protein was observed. In both systems there was a linear correlation between hepatic TG content and apoA-IV mRNA abundance (r2 = 0.8965). To establish whether the induction of apoA-IV impacted TG secretion, SREBP-1aTg mice were crossed with apoA-IV knock out mice (A4-KO). Based on the Triton block method, SREBP1aTg/A4-KO mice demonstrated a 24% reduction in hepatic TG secretion rate, relative to SREBP1aTg controls. The basis for the decrease in TG secretion was not due to decreased particle number, as apoB production was unaffected; however, negative stain electron microscopy revealed a 33% decrease in the abundance of large diameter (>120 nm) VLDL particles. To explore the impact of human apoA-IV overexpression, a recombinant apoA-IV adenovirus (AdA4) was developed. Mice infected with AdA4 demonstrated abundant hepatic expression of human apoA-IV, and a 50% increase in hepatic TG secretion rate, relative to the LacZ control. In addition, after only three days of expression AdA4 reduced liver TG content by 40%. Although again, apoA-IV expression did not impact apoB secretion, negative stain electron microscopy revealed a 1.8-fold increase in the abundance of large diameter VLDL particles. In conclusion, we demonstrate that in steatotic mice, hepatic apoA-IV expression increases TG secretion by promoting lipoprotein particle expansion, thereby dramatically reducing hepatic lipid burden without increasing atherogenic lipoprotein particle number.

Expression and clinical significance of apolipoprotein E in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a poor prognosis. Our previous proteomic analysis found apolipoprotein E (ApoE) protein to be up-regulated in the sera of patients with PDAC. In this study, we sought to confirm this finding and investigate the relationship between ApoE and PDAC. We measured ApoE expression in tissues from PDAC patients and normal controls (NC) by real-time PCR, western blot, and immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was applied to measure the levels of ApoE and carbohydrate antigen 19-9 (CA19-9) in the sera from patients with PDAC and NC. Real-time PCR and western blots showed that the ApoE mRNA and protein levels were up-regulated in PDAC tissues. The immunohistochemical results revealed that overexpression of ApoE was detected in 43 of 55 (78.2 %) PDAC cases and 3 of 20 (15 %) NC. High levels of ApoE were more likely in PDAC patients with advanced T status and TNM stages (p = 0.023 and p = 0.018, respectively). The ELISA results also confirmed that ApoE levels were elevated in the sera of PDAC patients. The sensitivity and specificity for distinguishing PDAC from NC were 76.2 and 71.4 %, respectively, for ApoE, 66.7 and 85.7 %, respectively, for CA19-9, and 81.0 and 85.7 %, respectively, for their combination. These results suggest that ApoE may be a potential PDAC-related biomarker and alone or in combination with other markers may provide additional information for the diagnosis and clinical management of PDAC.

Abstract 1150: Biomarkers discovery and racial disparity in hepatitis C-associated hepatocellular carcinoma.

Abstract Cancer health disparities represent a major public health concern in the US. Even when factors such as socioeconomic status, carcinogen exposure, and access to care are accounted for, disparities persist in the form of higher overall incidence rates and worse clinical outcomes for minority populations than for overall population. A case of point is the racial disparities in liver cancer-related mortality. Hepatitis C virus (HCV) is the most significant contributing factor in the development of hepatocellular carcinoma (HCC). In the US, African Americans (AAs) have twice the prevalence of HCV/genotype 1 infection, and develop HCC at more than twice the rate as Caucasian (CA) counterparts. African Americans are, however, less likely to respond to interferon-based therapy than CAs, and have considerably lower likelihood of receiving liver transplantation. While it is evident that viral infection with HCV is associated with the development of HCC, there are critical gaps in our understanding of the biological basis for this racial disparity. The aim of the current study was to define the molecular signatures of HCV disease progression in liver & tumor tissue samples obtained from AA & CA patients using 8-plex iTRAQ-based proteomics coupled with bioinformatics data analyses. The raw data were analyzed by the ProteinPilot v3.0 using the paragon algorithm. Searches were performed against a comprehensive database generated from SwissPort, Refseq, and Tremble protein sequences. The data were normalized for loading error & background correction. The proteins with confidence score > 90% and with at least 1 peptide of 95% identification confidence were used for further quality control & differential expression analysis. The quality control analysis was performed using pairwise correlation plots, boxplots, principal component analysis (PCA), and unsupervised hierarchical clustering. Supervised analysis was performed to identify differentially expressed proteins (DEP), where the relative protein expression values were compared between groups (Normal vs. Cirrhosis (CIR), Normal vs. HCC, CIR vs. HCC). Based on our experimental design, 787 unique proteins were identified. Of those, 32 were differentially expressed between normal, cirrhosis & HCC groups. Targets validation using real-time PCR (RT-PCR) or western blotting (WB) shows racially distinct alteration in the expression of certain targets. For example, the mRNA expression levels of TF were 2 and18-fold higher in CIR & HCC, respectively in AAs compared to CAs. Similarly, the expression of APOA1 mRNA levels was 7-fold higher in HCC of AAs compared to CAs. This trend was similar to their protein expression levels using WB. However, the level of HNF4α protein was down regulated in AAs compared to CAs. This indicates that HNF4α does not regulate TF & APOA1 expression in HCC of AA samples. Citation Format: Simon T. Dillon, Manoj K. Bhasin, Xiaoxing (Stella) Feng, David Koh, Sayed Salih Daoud. Biomarkers discovery and racial disparity in hepatitis C-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1150. doi:10.1158/1538-7445.AM2013-1150

ASSA13-03-17 b3-Adrenoceptor Activation Improves the Formation of Atherosclerosis Plaque in Aged ApoE-Deficient Mice

Objective To study the effects of β 3 -adrenoceptor (β 3 -AR) activation on atherosclerotic plaque development in aged apolipoprotein E-deficient (ApoE -/- ) mice. Methods Male ApoE -/- mice were fed with a high-fat diet, starting on 10 weeks of age. Starting from 36 weeks of age, mice were randomly treated with atorvastatin (10 mg · kg -1 ·d -1 , by gavage), the β 3 -AR agonist BRL37344 (1.65 or 3.30 μg·kg -1 , twice a week, by intraperitoneal injection), the β3-AR antagonist SR52390A (50 μg·kg -1 , twice a week, by intraperitoneal injection), or saline vehicle (twice a week, by intraperitoneal injection) for 12 weeks (n = 10 per group). A group of wild-type C57BL/6J mice receiving normal diet was included as a healthy control. At the end of the 12-week treatment period, serum concentration of lipids were measured. Atherosclerotic plaque in the thoracic aorta was examined using Hematoxy-eosin staining. The extent of fibrosis in the plaque was examined using Masson staining. Western blot was uesed to determine the ApoA-I and SR-BI protein expression. Real-time quantitative PCR was used to determine β 3 -AR mRNA in the white adipose tissue and the ApoA-I, ApoA-II and SR-BI mRNA expression in liver tissue. Results High-fat diet in ApoE -/- mice led to hyperlipidemia and the area of and the extent of fibrosis in atherosclerotic plaque (P 3 -AR mRNA and SR-BI mRNA were lower, ApoA-I and ApoA-II mRNA were higher in ApoE -/- mice (P -/- mice receiving vehicle). Treatment with the β 3 -AR agonist BRL37344 decreased serum concentration of triglyceride, total cholesterol and non-high density lipoprotein cholesterol. The treatment increased high density lipoprotein cholesterol, downregulated of ApoA-I, ApoA-II and SR-BI expression and upregulated the β 3 -AR mRNA expression (P -/- mice receiving vehicle). Similar to atorvastatin, BRL37344 treatment dose-dependently reduced the plaque area and collagen content (P -/- mice receiving vehicle at a dose of 3.30 but not 1.65 μg·kg -1 ). Treatment with the β 3 -AR antagonist SR52390A did not affect any parameters (P > 0.05 for all measures vs. ApoE -/- mice receiving vehicle). Conclusions β 3 -AR agonist impeded the progression of atherosclerosis in ApoE -/- mice, possibly through improvement of lipid profile, ApoA-I, ApoA-II and SR-BI mRNA.

Protein Kinase C Controls Vesicular Transport and Secretion of Apolipoprotein E from Primary Human Macrophages

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ε, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

Postprandial Changes in High Density Lipoproteins in Rats Subjected to Gavage Administration of Virgin Olive Oil

Background and AimsThe present study was designed to verify the influence of acute fat loading on high density lipoprotein (HDL) composition, and the involvement of liver and different segments of small intestine in the changes observed.Methods and ResultsTo address these issues, rats were administered a bolus of 5-ml of extra-virgin olive oil and sacrificed 4 and 8 hours after feeding. In these animals, lipoproteins were analyzed and gene expressions of apolipoprotein and HDL enzymes were assessed in duodenum, jejunum, ileum and liver. Using this experimental design, total plasma and HDL phospholipids increased at the 8-hour-time-point due to increased sphingomyelin content. An increase in apolipoprotein A4 was also observed mainly in lipid-poor HDL. Increased expression of intestinal Apoa1, Apoa4 and Sgms1 mRNA was accompanied by hepatic decreases in the first two genes in liver. Hepatic expression of Abcg1, Apoa1bp, Apoa2, Apoe, Ptlp, Pon1 and Scarb1 decreased significantly following fat gavage, while no changes were observed for Abca1, Lcat or Pla2g7. Significant associations were also noted for hepatic expression of apolipoproteins and Pon1. Manipulation of postprandial triglycerides using an inhibitor of microsomal transfer protein -CP-346086- or of lipoprotein lipase –tyloxapol- did not influence hepatic expression of Apoa1 or Apoa4 mRNA.ConclusionAll these data indicate that dietary fat modifies the phospholipid composition of rat HDL, suggesting a mechanism of down-regulation of hepatic HDL when intestine is the main source of those particles and a coordinated regulation of hepatic components of these lipoproteins at the mRNA level, independently of plasma postprandial triglycerides.

Curcumin Induces ABCA1 Expression and Apolipoprotein A-I-Mediated Cholesterol Transmembrane in the Chronic Cerebral Hypoperfusion Aging Rats

Cerebral hypoperfusion or aging often results in the disturbances of cholesterol and lipoprotein, which have been well depicted as a common pathological status contributing to neurodegenerative diseases such as vascular dementia (VaD) and Alzheimer's dementia (AD). The pathway of the liver X receptor-β (LXR-β)/retinoic X receptor-α (RXR-α)/ABCA1 plays a vital role in lipoprotein metabolism. Curcumin, a kind of phenolic compound, has been widely used. It has been reported that curcumin can reduce the levels of cholesterol in serum, but the underlying mechanisms are poorly understood. In this study, we evaluated the effects of curcumin on the cholesterol level in brain, vascular cognitive impairment and explored whether the mechanisms for those effects are through activating LXR-β/RXR-α and ABCA1 expression and apoA-I. With a Morris water test, we found that curcumin treatment could attenuate cognitive impairment. With HE and Nissl staining, we found that curcumin could significantly ameliorate the abnormal changes of pyramidal neurons. Meanwhile, the expression of LXR-β, RXR-α, ABCA1 and apoA-I mRNA and protein were increased in a dose-dependent manner after curcumin treatment. Interestingly, both serum HDL cholesterol and total cholesterol levels were statistically higher in the curcumin treatment group than those other groups. We conclude that curcumin has the ability to activate permissive LXR-β/RXR-α signaling and thereby modulate ABCA1 and apoA-I-mediated cholesterol transmembrane transportation, which is a new preventive and therapeutic strategy for cerevascular diseases.

Apolipoprotein E attenuates acute rejection of experimental renal allografts

Event Abstract Back to Event Apolipoprotein E attenuates acute rejection of experimental renal allografts Anna Zakrzewicz1*, Srebrena Atanasova1, Dariusz Zakrzewicz2, Gabriele Fuchs-Moll1, Sigrid Wilker1, Kathrin Petri1, Winfried Padberg1 and Veronika Grau1 1 Justus-Liebig-University Giessen, Department of General and Thoracic Surgery, Germany 2 Justus-Liebig-University Giessen, Department of Biochemistry, Germany Apolipoprotein E (ApoE) is a multifunctional protein, originally described in the context of lipoprotein metabolism and cardiovascular disease. More recently, anti-inflammatory functions of ApoE have been documented. In this study, we test the hypothesis that ApoE attenuates acute renal allograft rejection. The Brown Norway to Lewis rat strain combinations was used to investigate fatal acute rejection. In addition, Fischer 344 kidneys were transplanted to Lewis rats to study reversible acute rejection. Isograft recipients and untreated Lewis rats were used as controls. ApoE mRNA expression was quantified in intravascular leukocytes accumulating in the blood vessels of renal grafts and in graft tissue. ApoE protein levels were assessed in blood plasma. To test the protective potential of ApoE, recipients of Brown Norway kidneys were treated with ApoE-mimetic peptide. The number of different graft infiltrating leukocytes was evaluated by immunohistochemistry. Intravascular graft leukocytes and renal tissue obtained from animals undergoing reversible acute rejection expressed increased levels of ApoE mRNA, whereas during fatal rejection, ApoE expression remained unchanged. On the protein level, no changes in ApoE were seen in plasma. However, the local leukocytic ApoE expression could result in increased ApoE concentrations inside graft blood vessels. Treatment of allograft recipients with peptide reversed fatal rejection and significantly improved animal survival. Immunohistochemical analysis revealed reduced number of infiltrating T cells. Furthermore, reduced mRNA expression of granzyme B could be detected. ApoE plays a protective role in acute organ rejection. This effect is probably mediated by a reduced influx of cytotoxic T cells. Keywords: Apolipoprotein E, acute rejection, Kidney Transplantation, Cytotoxic T Cells, granzyme B, intravascular leukocytes Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Zakrzewicz A, Atanasova S, Zakrzewicz D, Fuchs-Moll G, Wilker S, Petri K, Padberg W and Grau V (2013). Apolipoprotein E attenuates acute rejection of experimental renal allografts. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00324 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Anna Zakrzewicz, Justus-Liebig-University Giessen, Department of General and Thoracic Surgery, Giessen, Germany, anna.zakrzewicz@chiru.med.uni-giessen.de Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Anna Zakrzewicz Srebrena Atanasova Dariusz Zakrzewicz Gabriele Fuchs-Moll Sigrid Wilker Kathrin Petri Winfried Padberg Veronika Grau Google Anna Zakrzewicz Srebrena Atanasova Dariusz Zakrzewicz Gabriele Fuchs-Moll Sigrid Wilker Kathrin Petri Winfried Padberg Veronika Grau Google Scholar Anna Zakrzewicz Srebrena Atanasova Dariusz Zakrzewicz Gabriele Fuchs-Moll Sigrid Wilker Kathrin Petri Winfried Padberg Veronika Grau PubMed Anna Zakrzewicz Srebrena Atanasova Dariusz Zakrzewicz Gabriele Fuchs-Moll Sigrid Wilker Kathrin Petri Winfried Padberg Veronika Grau Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Identification and Characterization of Cyclic AMP Response Element-Binding Protein H Response Element in the Human Apolipoprotein A5 Gene Promoter

The cyclic AMP response element-binding protein H (CREBH) plays important roles in hepatic lipogenesis, fatty acid oxidation, and lipolysis under metabolic stress. Here, we report CREBH as a novel regulator of human APOA5. Knockdown of endogenous CREBH expression via small interfering RNA resulted in the downregulation of human APOA5 mRNA expression in human hepatoma cells, HepG2. Sequence analysis suggested that putative CREBH response element (CREBHRE) is located in the human APOA5 promoter region and is highly conserved in both human and rodent. To clarify whether the human APOA5 promoter is regulated by CREBH, we analyzed the human APOA5 promoter region using a transient transfection assay and determined that transfection of CREBH induced human APOA5 promoter activity. Moreover, it was shown that CREBH directly regulated human APOA5 gene expression by binding to a unique CREBHRE located in the proximal human APOA5 promoter region, using 5′-deletion and mutagenesis of human APOA5 promoter analysis and chromatin immunoprecipitation assay. Taken together, our results demonstrated that human APOA5 is directly regulated by CREBH via CREBHRE and provided a new insight into the role of this liver-specific bZIP transcription factor in lipoprotein metabolism and triglyceride homeostasis.

Post-transcriptional nature of uremia-induced downregulation of hepatic apolipoprotein A-I production

Chronic kidney disease is associated with premature death from cardiovascular disease, which is, in part, driven by high density lipoprotein deficiency and dysfunction. One of the main causes of high density lipoprotein deficiency in chronic kidney disease is diminished plasma apolipoprotein (Apo)A-I level. Plasma ApoA-I is reduced in dialysis patients and hepatic ApoA-I messenger RNA (mRNA) is decreased in the uremic rats. This study explored the mechanism of uremia-induced downregulation of ApoA-I. Human hepatoma derived cells were incubated in media containing whole plasma or plasma subfractionation from normal subjects and patients with end stage renal disease pre- and posthemodialysis. Cells and culture media were isolated to measure ApoA-I protein and mRNA. ApoA-I promoter activity was measured using transfection with a luciferase promoter construct containing the -2096 to +293 segment of ApoA-I gene. Finally, effect of uremic and control plasma was assessed on ApoA-I RNA stability. Exposure to uremic plasma significantly reduced ApoA-I mRNA expression and ApoA-I protein production. These effects were reversed by replacing uremic plasma with normal plasma. Although no difference in ApoA-I promoter activity was found between cells exposed to uremic and normal plasma, uremic plasma significantly reduced ApoA-I RNA stability. Experiments using plasma subfractions revealed that the inhibitory effect of uremic plasma on ApoA-I mRNA expression resides in fractions containing molecules larger but not smaller than 30 kd. The pre- and postdialysis plasma exerted an equally potent inhibitory effect on ApoA-I mRNA abundance. Uremia lowers ApoA-I production by reducing its RNA stability. The inhibitory effect of uremic milieu on ApoA-I mRNA expression is mediated by non-dialyzable molecule(s) larger than 30 kd.

Emerging therapeutic agents to lower lipoprotein (a) levels

Recent epidemiological and genetic studies have suggested that lipoprotein (a) [Lp(a)] is a causal mediator of cardiovascular disease (CVD). There is now interest in evaluating Lp(a) as a therapeutic target. This review will summarize emerging therapeutic agents to lower Lp(a). Apheresis is the most efficacious method to lower Lp(a). Currently, there are no approved drugs to specifically lower Lp(a). However, recent data has demonstrated that Lp(a) can be significantly lowered, along with reductions in other apolipoprotein B-100 (apoB) containing lipoproteins, with antisense oligonucleotides to apoB, monoclonal antibodies to proprotein convertase subtilisin/kexin type 9, cholesterol ester transfer protein inhibitors, and thyromimetics. The farnesoid X receptor/fibroblast growth factor axis and interleukin-6 also influence Lp(a) levels and may be targets of therapy. Finally, specific apolipoprotein (a) [apo(a)] inhibitors apo(a) have been developed and reduce apo(a) mRNA and protein levels up to 86% without significantly affecting other lipoproteins. Lp(a) remains the last major lipoprotein disorder without any specific therapy. With the strong and accumulating data on its role as a causal risk factor for CVD, a rationale exists to develop novel agents to reduce Lp(a) and test the hypothesis that this will lead to reduced CVD events.

Aberrant Apolipoprotein E Expression and Cognitive Dysfunction in Patients with Poststroke Depression

Apolipoprotein E (ApoE) is associated with some diseases with cognitive function defect. The purpose of this study was to examine the influence of ApoE on poststroke depression (PSD) risk and to define objective markers for diagnosis. The cognitive function, serum ApoE, and peripheral mononuclear blood cell ApoE mRNA expression of patients with PSD were compared to age-matched control patients with stroke and healthy volunteers. Sixty-seven patients with stroke were selected according to the cerebral infarction diagnosis standard of the Fourth National Cerebrovascular Disease Conference and divided into a PSD group (28 patients, 43-76 years old) or a control stroke group (39 patients, 43-78 years old) using the Hamilton Rating Scale for Depression, and compared to 40 healthy volunteers (42-78 years old). Cognitive function was evaluated by analysis of event-related potentials (ERPs), while expression of ApoE mRNA was determined by quantitative reverse transcription-polymerase chain reaction and serum ApoE by ELISA. The latencies of ERP components N2 and P3 were prolonged, and the P3 amplitude was lower in the PSD group compared to the control stroke group and healthy controls (p<0.01). There were no significant group differences in N1 and P2 latencies (all p>0.05). The latency of N2 was positively correlated to the P3 latency in the PSD group (p<0.05). No associations were detected between P3 amplitude, expression of ApoE mRNA, and serum ApoE in the PSD group (all p>0.05). The ERP results indicated that patients with PSD were significantly slower at identifying a target stimulus, suggesting deficits in perception and/or cognitive processing. Peripheral expression of ApoE mRNA was lower in the PSD group than the control stroke group (p<0.701) while serum ApoE was higher than in the control stroke group (p<0.05), possibly reflecting a feedback reduction in expression. We suggest that aberrant serum ApoE together with abnormalities in some ERP components may be useful markers for assessment of PSD risk and clinical diagnosis.

Serum Apoe as a Reliable Marker to Monitor Graft Function after Hepatocyte Transplantation

Background: Hepatocyte transplantation is a promising treatment for patients with acute liver failure or metabolic liver diseases. However, major limitations are the lack of reliable noninvasive markers of graft function and the limited engraftment of transplanted cells. Apolipoprotein E (ApoE) is almost exclusively excreted by hepatocytes and is important for lipoprotein metabolism. The ApoE knockout mouse is severely hypercholesterolemic and develops premature atherosclerosis. We established a hepatocyte transplantation model in the ApoE knockout mouse evaluating both serum ApoE as well as lipoprotein profiles as marker of engraftment and function of tranplanted wildtype hepatocytes. Inflammatory injury of transplanted hepatocytes is recognized to be a major cause of initial graft loss. Using this model we also investigated whether pretreatment with Anakinra (anti-IL-1 antagonist) with and without methylprednisolone could improve engraftment of transplanted wildtype hepatocytes. Methods: ApoE-KO mice (B6.129P2-Apoetm1 Unc N11) were transplanted with wildtype hepatocytes isolated from C57BL/6 mice by collagenase perfusion. Controls were transplanted with ApoE-KO hepatocytes. A total of 6 x 106 hepatocytes were transplanted by three seperate intrasplenic injections. Animals were treated before transplantation and daily thereafter for 7 days with either 100 mg/kg/day i.p. Anakinra, alone or in combination with 15mg/kg/day i.p. methylprednisolone. Control mice received sham injections. Weekly blood samples were taken for lipoprotein analysis and quantification of serum ApoE by ELISA. Expression of ApoE mRNA in liver and spleen was quantified by TaqMan RT-PCR. Results: The low level of hepatocyte engraftment (1.5%) did not alter cholesterol or triglyceride lipoprotein profiles in ApoE-KO mice. Repeated hepatocyte transplantations significantly increased liver repopulation. Serum ApoE levels increased two-fold with each hepatocyte infusion. Serum ApoE levels correlated well with hepatic mRNA expression indicating that serum ApoE is a reliable marker of hepatocyte engraftment. Treatment with either Anakinra with and without methylprednisolone did not significantly increase serum ApoE or hepatic mRNA expression. Conclusions: Serum ApoE seems to be a sensitive and reliable marker of engraftment and function of transplanted hepatocytes. Blocking the inflammatory injury of transplanted hepatocytes by Anakinra with or without methylprednisolone does not significantly improve hepatocyte engraftment.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

The effects of berberine on hyperhomocysteinemia and hyperlipidemia in rats fed with a long-term high-fat diet

BackgroundThe study was undertaken to examine the effects of berberine (BBR) on serum homocysteine, lipids and the aortic lesion in Sprague–Dawley (SD) rats fed with a long-term high-fat diet (HFD).MethodsHealthy male SD rats weighing 190-210 g received randomly standard diet or a high-fat diet for 24 weeks. After 8 weeks of feeding, rats fed with HFD were randomized to receive berberine (200 mg · kg-1· day-1) or vehicle by gavage for 16 weeks. After overnight fasting, all rats were sacrificed and total blood samples were also collected for determinant of fasting serum homocysteine (Hcy), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-c) levels. The aorta was stained with hematoxylin and eosin (HE) and Sudan Ш to evaluate aortic lesion. The livers were dissected out and snap-frozen in liquid nitrogen for hepatic TC content and molecular analysis. 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), Lipoprotein receptors and apolipoproteins gene expression in the liver were determined by real-time PCR.ResultsIntragastrical administration with berberine for 16 weeks lowered serum Hcy in rats fed with a high-fat diet. In parallel, it also decreased body weight and improved serum TC and LDL-c. Berberine also tended to decrease hepatic cholesterol. Consistently, berberine also upregulated LDL receptor (LDLR) mRNA level and suppressed HMGR gene expression. Meanwhile, upon berberine-treated rats, there was a significant increase in apolipoprotein E (apoE) mRNA, but no change in apoAI and scavenger receptor (SR) mRNA in the liver. Further, no atherosclerotic lesions were developed in berberine-treated rats for 16 weeks.ConclusionBerberine can counteract HFD-elicited hyperhomocysteinemia and hyperlipidemia partially via upregulating LDLR and apoE mRNA levels and suppressing HMGR gene expression.

Tissue cholesterol content alterations in streptozotocin-induced diabetic rats

Diabetes is associated with elevated serum total cholesterol level and disrupted lipoprotein subfractions. The aim of this study was to examine alterations in the tissue cholesterol contents closely related to diabetic complications. Intraperitoneal injection of streptozotocin was used to induce type 1 diabetes in adult male Sprague-Dawley rats. On d 35 after the injection, liver, heart, intestine, kidney, pancreas, cerebral cortex and hippocampus were isolated from the rats. The content of total and free cholesterol in the tissues was determined using HPLC. The ATP-binding cassette protein A1 (ABCA1) protein and ApoE mRNA were measured using Western blot and QT-PCR analyses, respectively. In diabetic rats, the level of free cholesterol was significantly decreased in the peripheral tissues, but significantly elevated in hippocampus, as compared with those in the control rats. Diabetic rats showed a trend of decreasing the total cholesterol level in the peripheral tissues, but significant change was only found in kidney and liver. In diabetic rats, the level of the ABCA1 protein was significantly increased in the peripheral tissues and cerebral cortex; the expression of ApoE mRNA was slightly decreased in hippocampus and cerebral cortex, but the change had no statistical significance. Type 1 diabetes decreases the free cholesterol content in the peripheral tissues and increases the free cholesterol content in hippocampus. The decreased free cholesterol level in the peripheral tissues may be partly due to the increased expression of the ABCA1 protein.

Abstract 109: Role of ApoAIV in ApoB Lipoprotein Assembly and Triglyceride Transport

Apolipoprotein A-IV (apoAIV) is a lipid binding protein expressed primarily in mammalian intestine and also in rodent liver. While in vitro studies have suggested a role of apoAIV in lipoprotein formation and triglyceride (TG) transport, limited evidence supports such a function in vivo. To explore if hepatic apoAIV is uniformly regulated by increased hepatic TG levels, as is the case in the intestine, we studied three distinct mouse models of hepatic steatosis. These included high fat diet (HFD)-fed wild type mice, mice overexpressing a constitutive active form of SREBP-1a (SREBP-1aTg), and mice in which hepatic TG hydrolysis is blunted (mice treated with an anti-sense oligonucleotide (ASO) targeting knockdown of CGI-58/ABHD5). High fat diet-fed mice displayed a 4.5-fold increase in liver TG content and a 10-fold increase in apoAIV mRNA expression and protein mass. SREBP-1aTg mouse liver demonstrated an ∼35-fold increase in hepatic TG, apoAIV mRNA and apoAIV protein. A linear correlation was observed between hepatic TG content and apoAIV mRNA expression in the HFD fed wild type and SREBP-1aTg mice (r2 = 0.6852; p &lt; 0.01). Interestingly, while the TG content of CGI-58/ABHD5 knockdown animals increased by 4-fold, no corresponding increase in apoAIV expression was observed. To determine if the increase in apoAIV expression in the steatotic livers of SREPB-1aTg mice plays a role in the secretion of TG-rich apoB-containing lipoproteins, we crossed these mice with apoAIV knock out mice (A4-KO). The SREBP1a-Tg/A4-KO mice did not demonstrate a change in liver to body weight ratio or a change in hepatic TG content. Despite the large induction of apoAIV in the livers of SREBP1a-Tg mice, no difference was observed in hepatic TG production rates or VLDL particle size when apoAIV was deficient (SREBP1a-Tg/A4-KO). These results indicate that hepatic apoAIV expression is upregulated under conditions of enhanced lipogenesis, but is not altered under condition of defective TG hydrolysis. Furthermore, even under conditions of a 35-fold induction, apoAIV does not exert a detectable impact on TG transport or VLDL particle characteristics in mouse liver.

Abstract 493: Antiatherosclerotic Activity of a New P2y13 Receptor Agonist (ct1007900) in Animal Models

The F1-ATPase/P2Y13 receptor pathway has been involved in the regulation of the HDL uptake liver and disposition via the reverse cholesterol transport. CT1007900 is a novel selective P2Y13 receptor agonist that has been shown to enhance the HDL uptake that results in the increased secretion of the bile acid, bile cholesterol and bile phospholipid into the gallbladder in mice. In the present study, CT1007900 has been evaluated in three different animal models of atherosclerosis. In ApoE -/- flow cessation model, the administration of the drug decreased the cholesterol concentration in atherosclerotic plaques. In a high fat diet fed ApoE -/- mouse model, the prevention of the progression of the plaque in aorta was further evaluated after 4-week treatment with CT1007900. The treated animals had significant decreases in plaque area, cholesterol content, VCAM1 expression and macrophage content. In the high fat diet fed rabbits, 4-week treatment reduced the cholesterol content by about 30% and also decreased the thickness of the plaques. The ApoA1 mRNA levels in the liver and ApoA1 protein concentration in the plasma increased in the drug-treated animals. The HDL content of the treated animals showed a very consistent pattern with a specific decrease in large HDL and an increase of the “intermediate” size HDL particles as compared to control animals. These intermediate HDL particles seem to be more efficient particles for the removal of cholesterol from atherosclerotic plaques by increasing efflux of cholesterol from the macrophages present in the lesions. The plasma samples from drug treated rabbits showed a dose-dependent increase in the cholesterol efflux in an in vitro assay using J774 cells. These results clearly demonstrate that improving functionality of HDL rather than the levels of HDL could have a positive impact on the atherosclerotic pathology. These data also support that P2Y13 receptor agonists could be useful pharmacological therapeutics for the treatment of complications due to atherosclerotic disease.

TNF‐á inhibits stimulation of apolipoprotein A‐IV expression induced by linoleic acid (LA) in Caco2 cells

BackgroundChronic feeding of a HF diet significantly reduces intestinal as well as hypothalamic apolipoprotein A‐IV (apoA‐IV) gene expression by fat feeding. Since apoA‐IV suppresses food intake, this blunting of gut &amp; hypothalamic responses to fat feeding may contribute to diet‐induced obesity. It is well established that the circulating pro‐inflammatory cytokines are increased by HF feeding.AimThe following study was conducted to determine if pro‐inflammatory cytokines are involved in blunting the apoA‐IV responses to fat feeding.Results &amp; DiscussionsLA, an important dietary fatty acid, was used to stimulate apoA‐IV expression in human intestinal Caco2 cells. Cells were pre‐treated with or without human recombinant TNF‐á, IL‐6 or both before the addition of LA to stimulate apoA‐IV expression. ApoA‐IV mRNA levels were measured by real‐time RT‐PCR. LA stimulated apoA‐IV expression markedly in a dose and time dependent manner. Pre‐treatment with the cytokines for 48 or 72 h, especially with TNF‐á, significantly reduced the mRNA of apoA‐IV induced by LA.ConclusionsWe conclude that pro‐inflammatory cytokines, especially TNF‐á, are responsible for blunting of apoA‐IV response to fat feeding. Because of apoA‐IV's role in satiety, this interaction between circulating pro‐inflammatory cytokines and apoA‐IV may contribute to diet‐induced obesity. Supported by NIH DK 59630, DK 92138, and DK 76928.