Oral Presentations - Arteriosclerosis, Thrombosis, and Vascular ...

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A Role for C/EBP in Hepatic Transcription of the Gene Encoding TAFI Michael B Boffa, Jeffrey Hamill, Rebecca Dillon, Michael E Nesheim, Marlys L Koschinsky. Queen’s University, Kingston, ON, Canada P84 Thrombin activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that appears to play a role in regulating the balance between the activities of the coagulation and fibrinolytic cascades. Plasma concentrations of TAFI are largely genetically determined and vary in the population by almost an order of magnitude. Emerging data suggest that TAFI concentrations constitute a risk factor for thrombotic disorders and that TAFI is a positive acute phase reactant. Currently, no information exists concerning the mechanisms underlying control of TAFI gene expression. Using a computer algorithm to locate consensus transcription factor binding sites, we identified overlapping potential binding sites for the liver-enriched transcription factors C/EBP and hepatic leukemia factor (HLF) in the TAFI promoter. Based on the known sequence requirements for binding of the respective transcription factors, single nucleotide mutations were introduced into the TAFI promoter that would be expected to abolish either C/EBP or HLF binding or both. The mutant promoter sequences were used to construct luciferase reporter plasmids that were transiently transfected into HepG2 cells. Mutations that would be expected to abolish C/EBP binding alone or both C/EBP and HLF binding drastically reduced TAFI promoter activity (to 10 –20% of wild-type) while the mutation that would be expected to abolish HLF binding alone had no effect. Gel mobility shift assays and supershift assays revealed that this site was able to bind C/EBP present in nuclear extracts prepared from HepG2 cells and adult rat liver; C/EBP was the predominant binding isoform in the HepG2 nuclear extracts (consistent with the fetal-like phenotype of this cell line) while both C/EBP and C/EBP bound in the adult rat liver nuclear extracts. Importantly, binding site mutations that decreased TAFI promoter activity also abolished C/EBP binding. These results were corroborated using nuclear extracts from a non-hepatic cell line (Baby Hamster Kidney (BHK)) overexpressing C/EBP isoforms; these experiments also showed that C/EBP, which is induced in liver during the acute phase, also is capable of binding to the TAFI promoter C/EBP site. P85 Fabry Disease in Mice Is Associated with Age-Dependent Susceptibility to Vascular Thrombosis Peter F Bodary, Yuechen Shen, Susan R Wild, Akira Abe, James A Shayman, Daniel T Eitzman. University of Michigan, Ann Arbor, MI Fabry disease is an x-linked lysosomal storage disorder due to deficiency of -galactosidase A activity that results in widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction and stroke occur in patients with this condition primarily due to progressive deposition of glycosphingolipids in vascular endothelial cells. The clinical consequences of Fabry disease suggest that vascular thrombosis may play a prominent role in the pathogenesis of this disease, however the vasculopathy associated with Fabry disease has not been extensively studied. To determine if Fabry mice are susceptible to vascular thrombosis, mice deficient in -galactosidase A were studied in a photochemical thrombosis model. In this model of carotid artery thrombosis, Fabry mice displayed a progressive age dependent shortening of the time to occlusive thrombosis following vascular injury that correlated with progressive accumulation of the glycosphingolipid, Gb3, in the arterial wall (Figure). No age-dependent changes in thrombosis were observed in wild-type mice. Therapies designed to reduce the accumulation of Gb3 were also tested in this thrombosis model. Both substrate deprivation therapy and enzyme replacement therapy were ineffective in attenuating the prothrombotic response despite a reduction in vascular Gb3. These studies reveal a potent vascular prothrombotic phenotype in Fabry mice that is resistant to therapies designed to reduce Gb3 accumulation. P86 Antibody Inhibition of V and 3 Integrins but Not Inherited 3-Integrin-Deficiency Protects Mice from Developing Intimal Hyperplasia after Vascular Injury Susan S Smyth, Hsiming Yu, Barry S Coller, Ernane Reis. University of North Carolina, Chapel Hill, NC; The Rockefeller University, NY, NY; Mount Sinai School of Medicine, NY, NY Percutaneous coronary interventions are increasingly used to treat coronary artery disease, but restenosis still limits their utility. The 3 integrins (IIb3 and V3) have been implicated in events that may contribute to the development of intimal hyperplasia and restenosis after vascular injury. In a mouse model of endoluminal femoral artery injury in which endothelial denudation is followed by sequential platelet deposition and leukocyte accumulation, 3integrin-deficient mice are not protected from the development of intimal hyperplasia. Since inhibitors of V3(IIb3) reduce intimal hyperplasia after vascular injury in other animal models, we performed bilateral arterial injury in wild-type Downloaded mice; in wild-type from mice treated with http://atvb.ahajournals.org/ a control hamster F(ab)’ 2, an anti-mouse V F(ab)’ 2, or an anti-mouse 3 F(ab)’ 2; and in 3-/mice. Mice received antibody (2 mg/kg, ip) 1 h prior to surgery and then daily for 9 days. Intimal, medial and luminal areas were measured four weeks after injury. The mean intima/media ratio in the mice treated with anti-V or anti-3 F(ab)’ 2 were reduced by 60% compared to the control antibody-treated wild-type mice (see Table). The mean luminal area in the mice treated with antibody to V or3 was 1.5 times greater than the luminal area in untreated and control antibody-treated wild-type mice. The difference in response to arterial injury in mice lacking 3 integrins on an inherited basis and wild-type mice treated with inhibitors of 3 integrins may be due to upregulation of compensatory molecules in 3-/- mice or an effect of the anti-3 antibody on distinct integrins. Inhibition of Tumor Growth Using an Antisense Morpholino Oligomer to Integrin v Subunit Michelle A De Sousa, James D Bird, Carla A London, Patrick L Iversen, Gayathri R Devi, David H Farrell. Oregon Health & Science University, Portland, OR; AVI BioPharma, Corvallis, OR The microvascular endothelial cell integrin and fibrinogen receptor, v 3, is highly up-regulated during angiogenesis, which occurs during such processes as wound healing and neovascularization of tumors. Our laboratory is investigating the role of v 3 and its interaction with fibrin during angiogenesis, and studying possible anti-angiogenic therapies directed against v 3. Direct binding inhibitors of v 3 such as monoclonal antibodies and synthetic peptides have been used by other investigators to inhibit v 3 and prevent angiogenesis. Our approach involved the inhibition of angiogenesis by down-regulation of expression of the v 3 protein itself. This was achieved in a murine xenograft model by using a specific 20-mer phosphorodiamidate morpholino oligomer (PMO) directed against the mouse v mRNA. The PMOs represent a novel antisense structural type, wherein the deoxyribose moiety of DNA is replaced with a six-membered morpholine ring and the charged phosphodiester internucleoside linkage is replaced with a phosphorodiamidate linkage. The PMOs are neutral and avoid a variety of potentially significant limitations observed with the earlier generation of ionic phosphorothioate structural type oligomers. Nude mice were injected subcutaneously with human glioblastoma cell line U87MG. Established tumors were treated intratumorally with saline or 300 g antisense oligomer, or a scrambled version of the oligomer. Tumors of mice injected with the v antisense oligomer showed decreased average volume and mass compared to scrambled oligomer- and saline-injected controls. 50% of the test animals showed actual tumor regression. Those mice that showed tumor regression also had the lowest ratios of v to total protein, indicating that inhibition of v expression correlated with inhibition of tumor growth. These results suggest that the PMO antisense inhibition of v 3 gene expression is sequence-specific and causes inhibition of tumor formation in vivo. Regulation of PAI-1 Promoter Activity by SREBP Poster Presentations a-15 P87 P88 WITHDRAWN John A Schoenhard, Layton H Smith, Douglas E Vaughan. Vanderbilt University Medical Center, Nashville, TN Although HMG-CoA reductase inhibitors consistently improve endothelial-dependent vasodilation, their effects on other aspects of endothelial function are less well defined. Reduction of serum cholesterol levels and inhibition of intracellular isoprenylation are proposed mechanisms by which statins may limit plasminogen activator inhibitor-1 (PAI-1) expression and thus improve vascular fibrinolytic balance. In this study, we tested the alternative hypothesis that activated sterol response element binding protein (SREBP) may regulate PAI-1 expression. This was accomplished using PAI-1 promoter / luciferase reporter constructs in transfected endothelial cells. Overexpression of constitutively active SREBP1a induced -3.4 and -0.8 kb PAI-1 promoters by 11.5- and 9.5-fold, respectively (P 0.001). While further truncation of the PAI-1 promoter to -0.4 kb eliminated SREBP1a responsiveness, fusion of the distal PAI-1 promoter (-0.8 to -0.4 kb) to a minimal PAI-1 promoter (-52 to 76 bp) conferred novel SREBP1a inducibility to this construct, demonstrating that sequence elements located between -0.8 and -0.4 kb are both required and sufficient for SREBP1a mediated activation. Indeed, this region contains three potential SREBP binding sites, namely a consensus sterol response element (-766 to -757) and two E-boxes (-680 to -675, -569 to -564), the latter of which we have defined as responsive to other bHLH transcription factors including BMAL1, BMAL2, and TFE3. Mutation of these sites, however, failed to obviate SREBP1a induction of the PAI-1 promoter, indicating that activated SREBP may induce PAI-1 expression by an indirect or novel mechanism. Balanced against previous studies that have focused on mechanisms by which statins may limit PAI-1 expression independent of SREBP activation, our results suggest that SREBP activation may enhance PAI-1 expression, thus providing an explanation for inconsistent results obtained so far from clinical studies concerning the effects of statins on plasma PAI-1. Furthermore, our results suggest that lipid lowering therapies more narrowly targeted to SREBP activation by mayguest not capture on April potential 4, fibrinolytic 2013 benefits of statin therapy. P89
a-16 Arterioscler Thromb Vasc Biol. Vol 22, No 5 P90 Anti-Atherogenic Effect of Schistosoma Mansoni Infection in Apolipoprotein E Knockout Mice Christopher L Jackson, Ronald G Stanley, Michael J Doenhoff. Bristol Heart Institute, Bristol, UK; University of Wales Bangor, Bangor, UK OBJECTIVES Over 200 million people in the tropics are infected with schistosomes. Infections are heaviest during the first two decades of life and then generally decrease, but many people retain a residual worm burden into old age. The stable relationship that is eventually established between host and parasite in older people could be an adaptation by which the host derives some benefit from the parasite. In order to test this hypothesis, we have used an experimental animal model to test whether schistosomiasis has an effect on atherosclerosis. METHODS Male and female apolipoprotein E knockout mice were fed a diet containing 21% lard and 0.15% cholesterol from 8 weeks. Two weeks after initiation of high-fat feeding half of the animals were infected with 25 Schistosoma mansoni cercariae; the remainder of the animals were uninfected controls. Eighteen weeks after infection mice were terminated with perfusion exsanguination and fixation. RESULTS Control animals had typical lesions in the brachiocephalic arteries: large eccentric plaques containing large lipid cores with extensive cell death and destruction, covered by a fibrous cap. Atherosclerotic plaques in the brachiocephalic arteries of mice with 16 week patent schistosome infections were reduced in size by approximately 50% compared with uninfected controls (values given as mean/-SEM; control, n15, 165180/-25620 um2; infected, n14, 68080/-20820 um2: p0.05). The lesions consisted of a small lipid-rich region covered by a fibrous cap, with a necrotic core. Infected animals also had lower total plasma cholesterol concentrations (control, n15, 100/-5 mmol/L; infected, n14, 60/-4 mmol/L: p0.001). CONCLUSIONS Infection of apolipoprotein E knockout mice with Schistosoma mansoni decreases both plasma cholesterol concentration and atherosclerotic lesion size. Electrical Stimulation of Blood Vessels to Increase Tissue Plasminogen Activator Production: A Novel In Vivo Approach to Prevent and Treat Thrombosis Salah N Almohammed, Amer M Amer, Adam B Wienfeld, Saleh M Shenaq. Baylor College of Medicine, Houston, TX Objectives: Overcoming arterial and venous thrombosis presents a great challenge for physicians of all disciplines. Currently available therapeutic and preventive modalities range from aspirin to recombinant thrombolytic agents. Although these medications reduce the adverse impact of thromosis, untoward side effects remain a great concern. Tissue plasminogen activator (t-PA) is an effective anti- thrombotic peptide produced by the vascular endothelial cells. The aim of this study is to evaluate the effect of direct electrical stimulation (DES) on increasing t-PA level and activity for thrombus formation prevention and lysis. Material and Methods: Human endothelial cell culture has shown increased t-PA production after alternating current (AC) electrical stimulation. For the in-vivo studies, 31 NZW male rabbits were used to evaluate t-PA level after DES. The left femoral artery of each rabbit in the stimulation groups was cleaned, isolated and stimulated with 15 volts for 5 or 45 minutes using specific electrode design. Phosphated buffered saline aliquots were then ( 5 min., 24 hrs and 48 hrs after DES ) incubated into the lumen of the femoral artery and harvested after 30 minutes. t-PA level, activity and t-PA mRNA were then tested for using ELIZA, activity assay and rt-PCR, respectively. Arterial sections were also taken to test for tissue damage using light and electron microscopy. Results and Conclusion: In all DES samples, t-PA level, activity and mRNA were increased. This increase was detected immediately after stimulation and as late as 48 hrs after stimulation. Increasing the duration of DES from 5 min. to 45 min resulted in 2 fold increase in t-PA level and activity. No significant arterial wall damage was noticed using light and electron microscopy. Direct electrical stimulation has shown to increase t-PA level and activity in cell culture and in-vivo. Such encouraging results have led us to start the second phase of applying this novel local approach for the prevention and treatment of thrombosis. P92 N-3 Fatty Acid-Stimulated Degradation of Apoprotein B100 in Hepatic Cells Involves Lipid Peroxidation Meihui Pan, Arthur I Cederbaum, Christopher Cardozo, Kevin J Williams, Edward A Fisher. Mount Sinai School of Medicine, New York, NY; Thomas Jefferson University, Philadelphia, PA We have shown that polyunsaturated n-3 fatty acids (DHA or EPA) stimulate pre-secretory degradation of apoB100 in a post-ER compartment of hepatic cells, through a process involving PI3 kinase, but not proteasomes, lysosomes, or cell-surface re-uptake (Fisher EA et al. J Biol Chem 276:27855, 2001). To further explore the underlying mechanisms, we incubated rat hepatoma cells or primary hepatocytes with the n-3 fatty acid DHA complexed to BSA, while blocking candidate cellular mediators. Inhibitors of the major classes of intracellular proteases had no significant effects on DHA-stimulated apoB100 degradation. N-3 fatty acids are precursors to eicosanoids, but aspirin (which blocks their formation) had no effect. Interestingly, treatment of cells with the Ca chelator EGTA or BAPTA blocked DHA-stimulated apoB100 degradation. There was, however, strong evidence against a role for calcium. Chelation of intracellular Ca with BAPTA-AM, pharmacologic blockage of Ca channels, depletion of the ER-calcium pool, and increasing Ca influx with an ionophore each had no effect. Because EGTA also chelates Fe, and DHA is subject to Fe-dependent peroxidation, we examined the role of this lipid modification. 1) Desferroxamine (DFX), a chelator of Fe, the antioxidant vitamin E (VE), and the synthetic antioxidant BHT, each inhibited DHA-stimulated apoB100 degradation by 60%. 2) DHA incubation, compared to BSA, resulted in elevated cellular levels of TBARS (a standard index of lipid peroxidation), which were reduced by co-treatment with EGTA, DFX, VE, or BHT. 3) DHA treatment resulted in the formation of high molecular weight apoB complexes, Downloaded consistent withfrom the known ability of lipid P91 http://atvb.ahajournals.org/ peroxides to promote protein aggregation. Summary: DHA-stimulated degradation of apoB100 is a post-ER process involving PI3 kinase and also lipid peroxidation, leading to modifications of apoB100 and its destruction by a novel proteolytic mechanism. Decreased Modified Lipoprotein Uptake in Macrophages Lacking Both Scavenger Receptor A I/II and CD36 Vidya V Kunjathoor, Maria Febbraio, Lorna Andersson, Roy Silverstein, Mason W Freeman. Massachusetts General Hospital, Boston, MA; Weill Medical College of Cornell University, New York, NY The scavenger receptor family of multi-ligand binding proteins comprises 6 subgroups, termed SR-A through SR-F. The SR-A type I and type II receptors and CD36 (a member of the SR-B family) bind modified forms of low density lipoprotein (LDL) leading to cholesterol ester accumulation and macrophage foam cell formation. Mice lacking either SR-A or CD36 have been generated and both groups show diminished modified lipoprotein uptake. CD36 null mice have markedly diminished but not absent atherosclerotic lesion formation, whereas the effect on atherosclerosis of the loss of SR-A has varied with the strain of mice used in the analysis. To further characterize the importance of these receptors in lipid uptake and atherosclerosis and to assess compensatory mechanisms responding to their loss, we have crossed SR-A(I/II)null and CD36 null mice. The resulting double null mice appeared healthy and were derived in the expected Mendelian ratios. Serum cholesterol levels were not significantly different in wild ty pe (825 mg/dl) and double null mice (10325 mg/dl). Macrophages derived from the double knockouts, when stimulated with lipopolysaccharide produced equivalent amounts of pro-inflammatory cytokines as compared to wild type animals (TNF, wild type 5.5 0.36 ng/mg protein; double null 8.10.4 ng/mg protein and IL-6, wild type 42.80.55 ng/mg protein; double null 503.2 ng/mg protein, n3 mice per group). In contrast, a 96% dec rease in the degradation of acetylated LDL was measured in peritoneal macrophages taken from the SR-A/CD36 deficient animals as compared to wild type mice (wild type 27,2926,920 ng/mg protein, CD36-/- 6,817655* ng/mg protein, MSR-/- 1,7253 08* ng/mg protein, double null 786201* ng/mg; * indicates p0.05 versus wild type). This uptake was substantially less than that measured in either SR-A-/- or CD36-/- mice. Reduction in oxLDL degradation and foam cell formation were also measured. These data suggest that alternative scavenger receptors do not compensate for the loss of SR-A and CD36. The double null mice have been bred into the apoE null strain of C57BL/6 mice and studies on the effect of atherosclerosis are now in progress. P94 Apolipoprotein L-1 (apoL-1) Levels and the Frequency of an apoL-1 Allele are Increased in Patients with CAD and Hyperglycemia Timothy S Albert, Philippe N Duchateau, Samir S Deeb, Clive R Pullinger, Min H Cho, Mary J Malloy, John P Kane, B Greg Brown. University of Washington School of Medicine, Seattle, WA; Cardiovascular Research Institute, University of California, San Francisco, CA Objectives: The function of the recently described apoL family of apolipoproteins is still unclear. ApoL-1, a plasma protein member of this family, is found on VLDL and HDL particles and is elevated in high triglyceride (TG) states. We measured apoL-1 levels, and genotyped for the Lys166Glu apoL-1 polymorphism, in subjects enrolled in the HDL Atherosclerosis Treatment Study (HATS) in order to characterize this new apolipoprotein in a population with low HDL-cholesterol (HDL-C35 mg/dL) and CAD. Methods: ApoL-1 levels on- (TapoL-1) and off- (BapoL-1) therapy were quantified by ELISA in 137 of 160 pts enrolled in HATS, a 3-year angiographic trial of lipid lowering therapy and antioxidant vitamins. Regressions of BapoL-1 with baseline variables (age, fasting glucose (FG), hyperglycemia (HG diabetes or impaired FG (FG110–125 mg/dL)), body mass index, smoking or hypertension history, insulin, and lipid/apolipoprotein levels) and TapoL-1 with change in coronary stenosis (%S) were completed. The Lys166Glu variant of the apoL-1 gene was analyzed for by restriction fragmentation (n141) after our initial findings in order to assess for associations of the Glu allele with apoL-1 level and HG. Findings: ApoL-1 was positively and independently related to HG (p0.001) and VLDL-TG (p0.028) by multivariate analysis (multiple r0.37, p0.0001). ApoL-1 (meanSD) corrected for VLDL-TG was 50% higher in HG (n35) than normal glycemic (NG, n101) pts (15.113.2 vs. 9.98.1 g/mL, p0.008). No association between TapoL-1 and %S was seen (r0.05, p0.52). The Glu allele (frequency0.204) was not independently associated with apoL-1 level (p0.18), however, its frequency was 2-fold higher in HG (n33) than NG (n108) pts (0.318 vs. 0.167, 2 7.20, p0.007). Conclusions: ApoL-1 does not appear to be pro-atherogenic for the population as a whole. HG pts with low HDL-C and CAD, however, have independent elevations of apoL-1 and an increased frequency of a less common apoL-1 allele. These findings lead us to speculate that apoL-1 may play a unique role in diabetes and/or diabetic CAD, possibly contributing to the dyslipidemia of diabetes. P95 The LXR Ligand T0901317 Induces Severe Hypertriglyceridemia and Very Low HDL in the DB/DB Diabetic Mouse Jeffrey W Chisholm, Scott A Mills, Prabha N Ibrahim, Richard M Lawn. CV Therapeutics, Palo Alto, CA Liver-X-Receptor (LXR) ligands are currently under investigation as potential therapeutic agents for the treatment of low HDL common in both non-diabetic and diabetic humans. T0901317 (Tularik), a selective LXR ligand, has been shown to raise ABC1 and HDL levels in non-diabetic animal models while causing a moderate hypertriglyceridemia through increased SREBP1c expression. However, the effect of LXR ligands in a diabetic background have not been investigated. T0901317 was administered i.p. (50 mg/kg/d) for 12 days to diabetic DB/DB mice (7 wk old). Analysis of plasma lipids revealed that T0901317 treatment caused massive increases by in plasma guest total on April cholesterol 4, 2013 (3.4X) and triacylglycerols (17X) with an unexpected P93
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