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using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a significant (30–37%) decrease in levels of total triglyceride, cholesterol, LDL cholesterol and apoB in all 6 patients. Total plasma apoE concentration was reduced from 7.4 0.9 to 4.3 0.2 mg/dl (-38 8%, P 0.05), predominantly due to a decrease in VLDL apoE (3.4 0.8 vs 1.7 0.2 mg/dl; -42 11%) and IDL/LDL apoE levels (1.9 0.3 vs 0.8 0.1 mg/dl; -57 6%). VLDL apoE production was reduced from 3.82 0.67 to 2.26 0.42 mg/kg/day (-36 10%, P 0.057) and total plasma apoE production was significantly reduced from 4.67 0.39 to 3.04 0.51 mg/kg/day (-34 10%, P 0.05). VLDL and total plasma apoE residence times, and HDL apoE kinetic parameters were not significantly affected by drug treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r 0.99, P 0.001; r 0.88, P 0.05, respectively, n 6). Our results demonstrate that atorvastatin causes a pronounced decrease in VLDL apoE and total plasma apoE concentrations and a significant decrease in VLDL apoE and total plasma apoE rates of production in patients with combined hyperlipidemia. P244 Validation of 1 H MRS in the Measurement of Hepatic Triglyceride Content in Mice In Vivo Xiaobo Lin, Nobuhiro Sakata, Joel Garbow, Zhouji Chen, Gustav Schonfeld. Washington University in St. Louis, St. Louis, MO Magnetic resonance spectroscopy (MRS), a noninvasive modality, may be useful for repeated measurements of hepatic lipids over time, in vivo. The methylene proton ( 1 H) signals in 1 H MRS originate mainly from the mobile fatty acyl chains of tissue triglycerides. Respiratory gating is essential for correct placement of voxels. Hepatic lipid contents were measured (n6) both in vivo using MRS and biochemically by lipid extraction followed by enzymatic assay. MRS results were significantly correlated with triglyceride levels obtained by biochemical analysis (correlation coefficient, r 0.97, P 0.001). MRS was used to investigate diurnal changes of hepatic triglyceride levels in the apoB38.9 truncation-bearing mice (n6), generated by conventional gene-targeting in embryonic stem cells and the Cre-loxP system. These mice have a reduced capacity to export hepatic triglycerides. Preliminary MRS data indicate that the mean hepatic triglyceride level of heterozygous apoB38.9 mice was 1.5-fold higher than wild type controls (n6) (P 0.07). Food intake for the wild types and the heterozygotes was similar, with 61% (10.2 g of 17 g) consumed in the dark cycle but only 39% (6.8 g of 17 g) in the light cycle. Heterozygotes, but not wild types, tended to accumulate more hepatic triglycerides at 22:00 hours and 6:00 hours than at 12:00 hours. Thus, MRS appears to be a useful tool for measuring hepatic triglyceride levels in mice, in vivo. Structure and Function Studies of Human Apolipoprotein A-V P245 Robert O Ryan, Michael N Oda. Children’s Hospital Oakland Research Institute, Oakland, CA Studies have been conducted to characterize a newly discovered plasma apolipoprotein, human apolipoprotein A-V (apoA-V). Recent studies reveal an important role of apoA-V in maintenance of plasma triacylglycerol (TG) levels. In a murine setting, apoA-V transgenic animals display a threefold decrease in plasma TG while apoA-V null mice have a fourfold increase in plasma TG levels. The mechanism whereby apoA-V achieves this effect has not been determined although it is known to associate with high-density lipoproteins in rodent plasma. We have examined the structure and function of isolated recombinant human apoA-V expressed in E. coli. Spectroscopic studies and secondary structure analysis reveals that apoA-I possesses a high degree of alpha helix secondary structure. Lipid binding studies conducted with recombinant apoA-V reveal a functional ability to transform phospholipid bilayer vesicles into reconstituted high density lipoprotein complexes. Studies of apoA-V structural and lipid binding properties may provide insight into the molecular mechanism whereby this protein influences plasma triacylglycerol levels. Measuring Blood Viscosity: A Key Factor in Shear Stress and Atherogenesis Kenneth R Kensey, Anders Boss. Rheologics, Inc, Exton, PA P246 Background: Shear stress is a key regulator of endothelial function. Shear stress depends on blood viscosity, flow velocity and arterial diameter. Of these three variables, blood viscosity is the most susceptible to influence by exogenous factors such as smoking, lipid levels and diabetes and is a likely mediator of several well-recognized risk factors. In a normal population, blood viscosity in the upper quartile is associated with a 50% increased risk of cardiovascular event, increasing to a 300% in individuals with pre-existing vascular disease. Method: We used a new device, the Kensey Rheolog, to study blood viscosity in 58 healthy subjects at the AHA Scientific Sessions 2001. The Kensey Rheolog provides blood viscosity data over a physiological representative range of flow rate (shear rate) without the need for anticoagulants. We also studied the effect of regular blood donation in a group of nine healthy volunteers. Results: The measurements showed little variation at high shear. More individual variations occurred at low shear rate. Women had on average a lower blood viscosity than men (po .00009). We saw a change in blood viscosity at low shear in individuals with postprandial hypercholesterolemia, as well as a difference between the individuals with high and low HDL. The nine individuals who gave blood regularly over a five-week period demonstrated a progressive drop in blood viscosity. Conclusions: Blood viscosity varies considerably depending on flow rate with a much higher viscosity at low flow rates. Cholesterol, HDL levels and HTC clearly influence blood viscosity. Given the critical role of blood viscosity in determining shear stress and its relationship to recognized risk factors, blood viscosity is a key determinant of cardiovascular risk and its study with appropriate technology willDownloaded increase our understanding from of atherogenesis. http://atvb.ahajournals.org/ Poster Presentations a-43 P247 WITHDRAWN P248 The Second Window of Antiarrhythmic Effect Mediated by Adenosine A1 Receptor Agonist R-PIA on Ischemia-Induced Ventricular Tachyarrhythmias in Rats Guo Jin-Ai, Jing Chen, Dai Cheng-Xiang. No. 466 Hospital of People’s Liberation Army, Beijing, China Objective The aim of this study was to determine whether pretreatment with adenosine A 1 receptor agonist R-PIA 24 hours previous to coronary ligation has antiarrhythmic effects on ischemia-induced ventricular tachyarrhythmias in rat. Methods Eighty male Wistar rats were randomly divided into five groups(n16).Group A :normal saline 0.2ml i.c each rat; Group B : R-PIA 0.03mg.kg -1 i.c each rat; Group C: R-PIA 0.03mg.kg -1 i.c and DPCPX 0.2mg. kg -1 i.c each rat; GroupD: R-PIA 0.03mg.kg -1 i.c and L-NNA5mg.kg -1 i.p each rat; GroupE: R-PIA 0.03mg.kg -1 i.c each rat and 20min previous to coronary ligation, glibenclamide 0. 3mg.kg -1 i.v each rat. Sixteen rats in separate groups, 24 hours after pretreatment as above, were opened chest and subjected to 30 min of regional myocardial ischemia by ligation of the left anterior descending coronary artery followed by 120 min of reperfusion, and continually monitored and analysed left ventricular pressure curve and the rhythm and frequency of heartbeat by Cardio2 soft ware. Combining left ventricular pressure curve with electrocardiography, analysed and educed the frequency, onset time ,duration and total onset number of ventricular tachycardia and fibrillation. The experiment data were processed by SPSS, followed by the Fisher protected least significant difference test (q test) and Chi-square test. The null hypothesis was rejected at P0.05. Results One-way analysis of variance (ANOVA) express no significant difference in parameters of left ventricular function, the rate and rhythm of heartbeat between groups before myocardial ischemia (P0.05). In 30 minutes suffered ischemic insult, the frequency and duration of ventricular tachycardia and fibrillation in group R-PIA, compared with group normal saline , significantly reduced (P 0.01);Adenosine A1 receptor antagonist DPCPX and ATP-sensitive potassium channels antagonist glibenclamide could abrogate complately and partly respectively antiarrhythmic effect mediated by adenosine A 1 receptor agonist R-PIA pretreatment on ischemia-induced ventricular tachyarrhythmias, but nitric oxide synthesis inhibitor L-NNA couldn’t weaken the role of R-PIA. Conclusion This study suggested that adenosine A 1 receptor agonist R-PIA pretreatment could bring on antiarrhythmic effect on ischemia-induced ventricular tachyarrhythmias, and the role of R-PIA was partly resulted from ATP-sensitive potassium channels open through post-receptor mechanism. Nitric oxide didn’t appear to participate in triggering mechanism of above the role. P249 Exacerbated Atherosclerosis of Double-Mutant Lyst beige , LDLr-/- Mice Is Not Due Solely to Altered Macrophage Function Natalie K Schiller, Audrey S Black, Nobuhiko Kubo, William A Boisvert, Linda K Curtiss. The Scripps Research Institute, La Jolla, CA In previous studies, we found that LDL receptor-deficient mice (LDLr-/-) crossed with lysosomal-defective Lyst beige mice (Bg,LDLr-/-) had increased atherosclerosis compared to control. Further, it was found that macrophage (M) staining in Bg,LDLr-/- aortic root lesions was decreased and M distribution was limited to the lesion lumenal surface. In this study, we tested whether altered M function in Bg,LDLr-/- mice contributed to their exacerbated atherosclerosis. First, Bg,LDLr-/- M were evaluated in vitro for cellular migration, lipid accumulation and apoE production. Splenocytes isolated from Bg,LDLr-/- mice migrated better than LDLr-/- splenocytes through a Matrigel-coated membrane in response to MCP-1 (p 0.007). Bone marrow-derived M were cultured for 24 hours with or without 50 g/mL acetylated LDL (acLDL) to assess the accumulation of cholesteryl ester and free cholesterol as well as to quantitate apoE production. Lipid accumulation was similar between Bg,LDLr-/- and LDLr-/- M. ApoE production was significantly increased in the Bg,LDLr-/- M cultures (p 0.007), however this difference disappeared upon addition of acLDL. To determine whether these in vitro differences in M function affected atherosclerosis, 24 male LDLr-/- mice (6–8 weeks) underwent total body irradiation (10 gy) and reconstitution with either Lyst beige (Bg) or control bone marrow. The mice were fed a high fat diet for 16 weeks beginning 4 weeks after irradiation and reconstitution. Surprisingly, no differences were observed in aortic root lesion area or lipid accumulation within the aorta. Further, both MOMA-2-positive M staining and distribution in the aortic root lesions were similar between mice reconstituted with Bg or control bone marrow. Although functional differences in Bg,LDLr-/- M were observed in vitro, in vivo manifestations of these differences as assessed by bone marrow transplantation were unremarkable. This suggests that defective M function is not solely responsible for the exacerbated atherosclerosis of the Bg,LDLr-/- mice and that Lyst beige defects in other cell types such as smooth muscle cells may also be involved. P250 Apolipoprotein B, Small Dense LDL and Body Mass Index as Diagnostic Criteria for Familial Combined Hyperlipidemia: Results of a 5-Year Follow-Up Study Mario Veerkamp, Jacqueline De Graaf, Jan Hendriks, Pierre Demacker, Anton Stalenhoef. UMC Nijmegen, Nijmegen, Netherlands Introduction: Familial Combined Hyperlipidemia (FCH) is diagnosed by total cholesterol (TC) and/or triglyceride (TG) concentration above the 90th percentile. We have shown in 299 subjects of 32 families that the diagnosis FCH was inconsistent in 26% of the subjects over a five years period (1994–1999). These results emphasize the need for re-evaluation of the diagnostic criteria for FCH. Aim of the study: To evaluate whether apolipoprotein B (apo B) levels, small dense LDL and body-mass-index (BMI) improve diagnostic criteria for FCH. Methods: In by total guest 32 families, on April including 4, 2013 299 subjects, were studied in both 1994 and 1999. Apo
a-44 Arterioscler Thromb Vasc Biol. Vol 22, No 5 B (immunonephelometry, mg/l) and LDL subfractions (density-gradient-ultracentrifugation) were determined in both 1994 and 1999. LDL subfractions were described by a quantitative K-value in which a negative value of K reflects small dense LDL. A subject was defined ’truly’- FCH when diagnosed FCH in 1994 and/or 1999. Logistic discriminant analysis (under condition of equal assessment of misclassification with maximal sensitivity and specificity) was used to find the optimal cut-off point for apo B, K-value and BMI for prediction of ’truly’- FCH. Likelihood ratio tests were used to select between models. Results: In total 40% (n121) of the subjects were ’truly’-FCH. The ’truly’ affected status of a subject was best predicted by apo B levels 1200 mg/l, a K-value of -0.10 and BMI 25.0 kg/m2 . Both sensitivity and specificity were satisfactionally high. Sens. ranges from 65% to 77% while the spec. ranges from 75% to 80%. Multiple logistic regression analysis showed that apo B, K-value and BMI (all p0.05) independently contribute to better diagnostic criteria for ’truly’- FCH than compared to FCH based on TC and/or TG alone. Stepwise selection procedures showed that K-value and BMI were sufficient to predict ’truly’- FCH (adjusted odds ratios 0.24 (95% CI: 0.09–0.60) and 3.8 (95% CI: 1.6–9.9), respectively). Conclusion: Our results show that apo B, small dense LDL and BMI significantly improve the established diagnostic criteria for FCH, in which K-value and BMI are most predictive. P251 Cytotoxicity in J774 Macrophage Foam Cells Induced by Intracellular Cholesterol Accumulation Is Attenuated by Incubation with Apolipoprotein AI Ginny L Kellner-Weibel, Steven J Luke, George H Rothblat. The Children’s Hospital of Philadelphia, Philadelphia, PA Excess intracellular free cholesterol (FC) is cytotoxic. The present study examines prevention of FC-induced cytotoxicity in J774 macrophage foam cells by incubation with apolipoprotein AI (apoAI). J774 macrophage cells were enriched with esterified cholesterol (EC) by exposure to acetylated low-density lipoprotein and FC/phospholipid dispersions, thus creating a model foam cell. Treatment of these foam cells with an acyl coenzyme-A:cholesterol acyltransferase (ACAT) inhibitor, CP-113,818 (2g/ml), in the absence of an extracellular cholesterol acceptor, resulted in hydrolysis of stored EC and subsequently FC-induced cytotoxicity. Incubation of foam cells with the ACAT inhibitor plus apoAI (50g protein/ml) resulted in FC efflux (0.22 0.02% / hour) along with a reduction in cytotoxicity (14.6 1.3% reduction), measured by adenine release. Small unilamellar vesicles (SUV, 100g phospholipid/ml) caused greater FC efflux (0.29 0.02% / hour), although only a modest reduction in cytotoxicity (4.7 3.0% reduction) was measured. Previously we have shown that the intracellular cholesterol transport inhibitor, U18666A abolishes FC-induced cytotoxicity when macrophage foam cells are incubated with the ACAT inhibitor by preventing movement of FC to the plasma membrane. In the present study, co-incubation of the ACAT inhibitor plus U18666A (2g/ml) reduced efflux to apoAI (40.6 1.31% reduction), but did not result in a reduction of efflux to SUV. Pre-treatment of the J774 mouse macrophage foam cells with CPT-cAMP (0.3mM for 12 hours) upregulates hormone sensitive lipase and ABCA1. In experiments using mouse serum as a cholesterol acceptor, CPT-cAMP caused greater protection against FC-induced cytotoxicity compared to cells without the pre-treatment, suggesting a role of ABCA1 in removal of cytotoxic FC. We conclude that in this system, a cytotoxic pool of FC is located in the plasma membrane, this FC is readily available for efflux to apoAI, and removal of excess cytotoxic cholesterol may involve the ABCA1 transporter. P252 Extracellular Superoxide Dismutase Gene Polymorphism in Mouse Models of Atherosclerosis Anson P Pierce, Jason Whitlark, Ladislav Dory. University of North Texas Health Science Center at Fort Worth, Fort Worth, TX We report for the first time a polymorphism in the mouse extracellular superoxide dismutase (ecSOD) gene. Two amplicons corresponding to the 3’ untranslated region of the ecSOD gene were detected in liver and macrophage cDNA from B6.129 ApoE and LDLR double KO mice by RT-PCR. Sequence analysis revealed that the two amplicons, reproduced by PCR from genomic DNA, differ by a 10 bp deletion in the 3’ untranslated region (bp 758–767). Restriction enzyme analysis revealed an additional single nucleotide polymorphism (SNP) site, an A-G change at bp 61, which always accompanies the 10bp deletion. This SNP leads to an Asn-Asp substitution that resides in the putative signal sequence of the protein. Since the B6.129 mouse is an F2 hybrid of the 129Sv/J and C57Bl/6J mouse strains, the 129P3/J (parental strain) and C57Bl/6J mice were genotyped, by a procedure developed in our lab, for the 10 bp deletion and the SNP. The atherosclerosis-resistant 129P3/J mouse is homozygous for the ecSOD gene containing the A-G change and the 10bp deletion (short gene), while the atherosclerosissusceptible C57Bl/6J mouse is homozygous for the longer variant of the ecSOD gene. This variation appears to influence the extent of ecSOD expression/activity. Thus, analyses of ecSOD activities show that age-matched female 129P3/J mice (athero-resistant) have 2-fold higher circulating ecSOD in plasma (p0.0042), 1.3-fold higher heparin releasable ecSOD (p0.0002), and a higher level of ecSOD activity bound to the arterial wall when compared to C57Bl/6J females (athero-susceptible). Age- and sex-matched B6.129 apoE and LDLR double KO mice homozygous for the long ecSOD gene have 2.6-fold lower ecSOD activity than heterozygous sex-matched littermates. A failure to recognize this polymorphism has led some investigators to incorrectly characterize the pattern of ecSOD expression during the development of atherosclerosis in mice. Therefore, studies using genetically altered, but insufficiently backcrossed, mouse models of disease should be Downloaded interpreted withfrom caution. http://atvb.ahajournals.org/ P253 Integrin-Dependent Adhesion Targets the Protein Synthetic Machinery of Platelets to Distinct Intracellular Regions: Evidence for the Development of a “Translational Core” Stephan Lindemann, Neal D Tolley, Guy A Zimmerman, Andrew S Weyrich. Human Molecular Biology and Genetics, Salt Lake City, UT Here we demonstrate that adherence to purified collagen, fibrinogen, and laminin markedly increases protein synthesis by platelets. As measured by incorporation of [ 35 S]-labeled proteins, proteins are differentially synthesized on each matrix, with fibrinogen yielding the most robust response. We next determined if the protein synthetic machinery is redistributed in fibrinogenadherent platelets and whether spatial targeting of this machinery is required for efficient translation of mRNAs. As expected, fibrinogen-adherent platelets form actin stress cables and vinculin-rich focal adhesion complexes. Platelet RNAs, ribosomes, and eukaryotic translation factors such as eIF4E (eukaryotic Initiation Factor 4E) and eIF2 (eukaryotic Initiation Factor 2) are redistributed to the middle of the cell, forming a nucleus-like complex that is surrounded by the actin stress cables, referred to here as the Translational Core. This translational core also contains 3-integrins, suggesting that integrins target the protein synthetic machinery to distinct regions within the cell. Inhibition of cellular adherence by IIb 3 inhibitors or cell spreading by cytochalasin D prevented the formation of the translational core and subsequent protein synthesis. Biochemical separation of translational components and subsequent cDNA array analysis revealed that adherence to fibrinogen markedly increases the number of transcripts bound to the mRNA-binding protein eIF4E compared to resting platelets. Two of these eIF4E-bound mRNAs included Bcl-3 and IL-1, proteins that are synthesized in an integrin-dependent fashion. Lastly, we demonstrate that 3 integrins and eIF4E are redistributed from the monosomes of quiescent cells to the polysome-rich fractions of fibrinogen-ligated platelets. Neutralization of IIb 3-integrins blocks this response. These data indicate that integrins target the protein synthetic machinery to the translational core of platelets and thereby regulate protein synthetic responses. P254 Baboon Lp(a) Does Not Bind to Lysine and Fibrin despite the Presence of a Strong Lysine Binding Site in Apo(a) Kringle IV Type 10 Janet Ho, Andrea Belczewski, Michael B Boffa, Marlys L Koschinsky. Queen’s University, Kingston, ON, Canada Insight into the role of lipoprotein(a) (Lp(a)) in fibrinolysis may be gained through comparative analysis of the structure of the distinguishing protein component, apolipoprotein(a) (apo(a)), from humans and other species. Human apo(a) contains ten types of plasminogen kringle (K) IV-like sequences, followed by sequences which are similar to the kringle V and protease domains of plasminogen. Human apo(a) KIV10 contains a strong lysine-binding site (LBS) which has been proposed to mediate the lysine-dependent interaction of apo(a) with biological substrates such as fibrin. Mutations in amino acids which form the LBS in KIV10 are present in both rhesus monkey (Trp72/Arg) and chimpanzee apo(a) (Asp57/Asn) and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. The objective of our study was to characterize the lysine-binding properties of Lp(a) in the baboon in order to further the comparative analyses with the other primate species. We sequenced a segment of baboon liver cDNA spanning from KIV9 to the protease domain. The organization of this region is similar to the rhesus monkey in that baboon apo(a) also lacks a KV domain. Interestingly, we found that the baboon KIV10 sequence contained all of the canonical residues identified in formation of the LBS. We then characterized the apo(a) KIV10 sequence from an additional 10 unrelated baboons; the sequence was identical to the initial clone except that one allele from each of two animals encoded Arg at position 72 rather than Trp. Based on the amino acid sequence of baboon KIV10, we were surprised to find that none of the corresponding Lp(a) species bound to lysine-Sepharose even upon partial purification. Additionally, we found that the Lp(a) bound very poorly if at all to plasmin-modified fibrin compared to human Lp(a). Expression and purification of baboon and human KIV10 in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that elements of apo(a) structure besides KIV10 sequence dictate the lack of lysine binding of baboon Lp(a), and perhaps that of other non-human primates. P255 Changes in Microvascular Structure and Growth Factor Expression in an Angiogenesis Model Eric M Brey, Larry V McIntire, Carol Johnston, Charles W Patrick Jr.. Rice University, Houston, TX; M.D. Anderson Cancer Center, Houston, TX Understanding the microarchitecture and expression of growth factors during microvascular development is an important aspect of both normal tissue physiology and the evolution of many pathologies. We previously reported the development of imaging methods that allow high-resolution three-dimensional (3D) imaging of vascular microstructure and automated analysis of comparative growth factor levels from immunohistochemistry. These methods were employed to quantify the spatial and temporal aspects of angiogenesis in a fibrin gel, a model of both wound healing and tissue engineering. Gels were implanted on a uniform muscle surface within Lewis rats. Explants were removed at 7 and 14 days and snap frozen. Forty serial 6-micron sections were cut on a cryostat, stained for CD31, digitally imaged, processed, and volume rendered for 3D images of vascular microstructure. Vessel parameters were calculated for the networks at each time point. Additional 6 mm sections were immunostained for specific growth factors: VEGF, bFGF, TGF-b1, PDGF-B, and Ang-2, and for H&E’s. These were selected due to their reported importance in the initial phase of angiogenesis and the subsequent maturation of vessels through the regulation of stability and recruitment of support cells. Growth factor levels were determined from the optical density of the samples. Statistical analysis was performed using ANOVA with Tukey-Kramer post test. Data are presented as mean /-by SEM. guest P0.05 on was April considered 4, 2013 statistically significant. Angiogenesis occurs rapidly
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