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with cytochalasin D reduced the movement of PDGFR-beta aggregates. The motility of PDGFR-beta aggregates also depended on the activity of PDGFR-beta tyrosine kinase, Rho kinase, and myosin light chain kinase. This study suggests that the movement of PDGFR-beta aggregates may facilitate cell membrane extension and thereby cell migration. Is apo B 48 Important for Chylomicron Metabolism? Cam Phan, Trevor Redgrave, Shuqin Zheng, Shrikant Anant, Nicholas O Davidson, David Y Hui, Patrick Tso. University of Cincinnati, Cincinnati, OH; University of Western Australia, Perth, WA, Australia; Washington University, St. Louis, MO P209 Apolipoprotein B 48 (apo B 48) is an intestinally derived apolipoprotein associated with chylomicrons. Chylomicrons are rapidly metabolized to form remnant particles before removal by the liver. In contrast, hepatic derived apo B 100 containing VLDL are converted to LDL. Is the difference in chylomicron and VLDL metabolism a result of the presence of apo B 48 versus apo B 100? The apobec-1 knockout mouse allows us the opportunity to test this possibility since the apoB mRNA editing enzyme, apobec-1 is not present and only apo B 100 containing chylomicrons are produced. In this study, chylomicrons were collected from C57BL/6 and from apobec-1 knockout mice and the plasma clearance of the different chylomicrons were studied in C57BL/6 mice. Lymph was collected from donor lymph fistula mice (apobec-1 or C57BL/6) infused intra-duodenally with an Intralipid/taurocholate mixture labeled with 14 C-cholesterol and 3 H-triolein. Chylomicrons were isolated by density gradient ultra-centrifugation and injected, via tail vein into recipient C57BL/6 mice. Blood samples were taken over 20 mins and plasma counted to determine the disappearance of both labels over time. Liver and spleen were removed at the end of 20 mins to determine label uptake. Chylomicron lipolysis was determined by the plasma disappearance of 3 H-triolein while chylomicron remnant removal was determined by the disappearance of 14 C-cholesterol. Plasma chylomicron-triolein and cholesterol disappearance curves were not different for chylomicrons produced from apobec-1 mice compared with those from C57BL/6. Within 3 mins post-injection of both types of chylomicrons, approximately 70% of the triolein label (chylomicron hydrolysis) disappeared from the circulation and 90% by 5 mins. About 35–40% of the cholesterol label (remnant removal) was removed by 3 mins and 90% by 20 min. Organ uptakes of both labels are also similar between wild type and apobec-1 knockout chylomicrons. We conclude: 1) the substitution of apo B 100 in place of apo B 48 in chylomicron particles had minimal effects on the plasma clearance of triglyceride and cholesterol; 2) the difference in plasma clearance between chylomicron and hepatic VLDL is not due to the type of apo B present. P210 Retinoids Activate Cyclic-AMP Response Element Binding Protein (CREB) in Vascular SMC via Protein Kinase A Jeffrey W Streb, Mary A Georger, Joseph M Miano. University of Rochester Medical Center, Rochester, NY Retinoids have been shown to inhibit smooth muscle cell (SMC) growth in vitro and in vivo. While the mechanism of this inhibition remains unclear, retinoids are known to act through the retinoid receptors, a group of ligand-activated transcription factors, to modulate gene expression. One such gene is the tumor suppressor SSeCKS, a multivalent scaffold protein that binds and modulates the activity of Protein Kinase A (PKA) and Protein Kinase C (PKC). A downstream target of both of these kinases is CREB, a multifarious transcription factor that also acts as a co-factor for the retinoid receptors. We therefore examined the possible role of CREB in mediating retinoid effects in SMC. Agonists that stimulate the cAMP-PKA-CREB pathway inhibited serum-induced SMC growth to a similar degree as retinoids. While forskolin raised global cAMP levels, retinoid treatment had little or no observed effect. However, levels of the phosphorylated, active form of CREB (pCREB) did increase following retinoid treatment. Further, retinoid treatment led to a dose-dependent increase in CREB-mediated transcription. In contrast to agonists that lead to a rapid, short-term elevation of pCREB and CREB-assisted transcription, the retinoid stimulated increase in pCREB and CREB-mediated transcription was protracted. The increase in pCREB was not due to an increase in total CREB as assessed by Western blotting. Since CREB can be activated via PKA- and PKC-mediated phosphorylation, we examined the role of these kinases in retinoid-induced CREB activation. Only the PKA inhibitor H-89 blocked forskolin-and retinoid-induced activation of CREB. These results indicate that retinoid-induced activation of CREB involves PKA, but is independent of global increases in cAMP. Since CREB can inhibit SMC growth, our results suggest a role for this transcription factor in retinoid-induced SMC growth inhibition. Further studies are underway to better define this pathway. FGF2 Overexpression Prevents Ceramide-Mediated Akt Deactivation in Endothelial Cells Exposed to Oxidized LDL Jonathan Lu, Shinichi Suzuki, Hazim J Safi, Wei Jiang, Philip D Henry, Chao-Yuh Yang, Chu-Huang Chen. Baylor College of Medicine, Houston, TX; University of Texas-Houston Medical School, Houston, TX P211 Apoptosis of vascular endothelial cells (EC) plays a critical role in atherothrombosis and modified lipoproteins are considered proapoptotic. We recently demonstrated that copperoxidized LDL (oxLDL) induces EC apoptosis in part by downregulating fibroblast growth factor 2 (FGF2). FGF2 is a primary activator of the phosphatidylinositol-3-kinase (PI3K) that activates Akt/protein kinase B by means of phosphorylation. Akt inhibits apoptosis by deactivating downstream apoptotic mediators. Because our primary experiments showed that oxLDL increased turnover of ceramide, an intracellular lipid mediator, we investigated the interaction between the FGF2-PI3K-Akt axis and ceramide in the signaling pathways triggered by oxLDL. In bovine aortic EC (BAEC) cultures, oxLDL (50 g/mL) induced marked apoptosis at 24 hours, as assessed by epi-fluorescence microscopy, DNA laddering, and flow cytometry. The apoptosis was accompanied by Akt deactivation, Downloaded as evaluated byfrom reduced phosphorylation. http://atvb.ahajournals.org/ Poster Presentations a-37 Wortmannin (50 nM), a PI3K inhibitor, also deactivated Akt and provoked oxLDL-like apoptosis. Fumonisin B-1 (400 nM), a ceramide synthase inhibitor, effectively attenuated oxLDL-induced Akt deactivation and the associated apoptosis, suggesting a participating role of newly synthesized ceramide. Both membrane permeable C2- and C6-ceramide (50 M) inhibited Akt phosphorylation and induced apoptosis. Cells transfected with adenoviral vectors containing an FGF2 antisense oligonucleotide completely abolished FGF2 expression and Akt phosphorylation. In contrast, overexpressing FGF2 in BAEC by transfecting the cells with FGF2 sense-containing vectors prevented oxLDL or ceramide-induced Akt deactivation and the associated apoptosis. Thus, oxLDL acts in part by stimulating production of ceramide, which participates in Akt deactivation. Concomitant FGF2 downregulation deprives the cells of an important defensive mechanism that relies on the integrity of the FGF2-PI3K-Akt axis. FGF2 overexpression renders the cells resistant to ceramide-mediated Akt deactivation in EC exposed to modified lipoproteins, such as oxLDL. P212 Topology and Functional Analysis of the Large Extracellular Aminoterminal and Regulatory Domains Michael L Fitzgerald, Lorna P Andersson, Sarah Rhee, Armando J Mendez, Mason W Freeman. Mass. Gen Hospital/Harvard Medical School, Boston, MA; University of Miami Medical School, Miami, FL Mutations in ABCA1 can result in a loss of cholesterol efflux from cultured fibroblasts and are responsible for causing Tangier disease. Hydrophobicity plots of ABCA1 predict more hydrophobic domains than the canonical 12 which would be expected in a full ABC transporter. Thus, the topological orientation of the ABCA1 polypeptide chain remains uncertain. Previously we have shown that the first hydrophobic domain of the protein (AA 25–42) acts as a signal anchor sequence that results in the translocation of the downstream 590 amino acids to the extracellular space. Here we report that the central highly hydrophobic domain (AA 1351–1372, or H8) also traverses the plasma membrane leading to the extracellular positioning of residues 1352–1649. Using the full length transporter with a FLAG tag epitope placed between AA 1396 and 1397 (Flag2), we found comparable antibody binding to this tag compared to the same tag placed in the amino terminal loop previously demonstrated to be extracellular (Flag1). Constructs expressing the H8 domain in the context of the entire central regulatory region (AA 843-1650), or a fragment of that region (AA 1311–1650), resulted in the production of a glycosylated protein, indicating that the H8 hydrophobic residues are capable of directing translocation across the lipid bilayer as opposed to serving as a hairpin insertion into the membranes as has been previously proposed. In 293 cells transfected with wild type ABCA1 or Flag1or Flag2, similar amounts of cellular cholesterol was effluxed to ApoA-I (wt 4.2 1.1 % v.s. Flag1, 4.6 0.6 % & Flag2 4.3 1.1 %). Cellular binding of ApoA-I was also similar (ng of specific ApoA-I bound/mg of cellular protein, wt, 9.31 0.63 v.s. Flag1, 9.1 1.4 & Flag2, 5.2 0.98). Both Flag proteins could be cross-linked to A-1 and the apoprotein immunoprecipitated with anti-ABCA1 antibody. These studies indicate that ABCA1 has extracellular aminoterminal and central regulatory loops and that the insertion of Flag tags in these loops is structurally compatible with retained ligand binding and efflux function of the transporter, making such tags useful in future cell biological investigations. P213 The In Vivo Performance of Vascular Grafts Cryopreserved by Vitrification K G M Brockbank, Y C Song, M J Taylor. Organ Recovery Systems, Inc., Charleston, SC The primary objective of our research program is the development of storage methods for living natural and tissue engineered arterial bypass grafts. It is well known that a principal problem in attempting to cryopreserve tissues and organs is formation of extracellular ice which destroys both tissue structure and function. Vitrification is an alternative approach to preservation that either completely avoids freezing or reduces ice crystallization to a tolerable minimum. Here we report the results from our 28 day in vivo studies employing autologous and allogeneic, vitrified, rabbit jugular veins. Patency was equivalent in the experimental and control groups. The in vivo response was assessed after placing the veins as bypass grafts in carotid arteries, followed by histopathology and smooth muscle physiology studies of elective explants. Our results demonstrate that the integrity of a multi-cellular system can be preserved in the vitreous state without the inherent problems associated with crystallization and so-called “solution effects injury” that arise in cells due to the removal of water during ice formation. Glass stability and cell viability of vitrified samples were retained during vapor phase liquid nitrogen storage for at least four months. Histopathology of fresh and vitrified veins demonstrated that the proliferative and remodeling responses of the vitrified grafts were normal. In addition, both endothelial cell and smooth muscle cell functions of vitrified specimens were well preserved post-transplantation relative to untreated control grafts. These studies suggest that the vitrification process had no discernible adverse effects on tissues assessed by transplantation in the rabbit. Fibrin-Associated Characteristics of Atheroma-Targeted Echogenic Immunoliposomes Melvin E Klegerman, Andrew J Hamilton, Susan D Tiukinhoy, Amer A Khan, Shao-Ling Huang, Robert C MacDonald, David D McPherson. EchoDynamics, Inc, College Park, MD; Northwestern University, Chicago, IL; Northwestern University, Evanston, IL P214 Antibody (Ab)-targeted echogenic immunoliposomes (EGIL) have been developed as novel ultrasound contrast agents for evaluation of vasoactive and pathological endothelium and atherosclerosis components, particularly surface-accessible fibrin in late-stage and vulnerable atheroma. In order to monitor the antibody targeting aspect of EGIL, methods have been developed to assess the immunoglobulin (Ig)-liposome (lp) conjugation (conj) efficiency routinely, but a reproducible, convenient method for quantitative assessment of EGIL binding to target antigens by guest is required. on April Previously, 4, 2013a radioimmunoassay (RIA) protocol was used to
a-38 Arterioscler Thromb Vasc Biol. Vol 22, No 5 establish that a minimum avidity vs. soluble human fibrinogen (fgn) of 1x10 12 M -1 /lp for EGIL conj to a polyclonal anti-human fgn/fibrin Ab (PAb) was required for optimal EGIL binding to fibrin in a physiologic flow simulator. Our objective was to develop a more convenient enzyme-linked immunosorbent assay (ELISA) procedure for routine assessment of EGIL targeting efficiency and to compare the results with the RIA data. Wells of polystyrene microtiter plates were coated with human fgn, half of which were then converted to fibrin with thrombin in order to evaluate the fibrin specificity of PAb-EGIL. Primary incubation of samples diluted in detergent-free buffer allowed determination of dissociation constants (K D) for intact EGIL. Secondary incubation with anti-IgG conj to alkaline phosphatase was followed by incubation with p-nitrophenylphosphate and measurement of absorbance at 405 nm (A 405). The average K D for PAb-EGIL binding to fgn at 37 o C, determined by RIA, was 1.77 nM, while the analogous ELISA value was 1.80 nM. The ELISA-determined K D for binding to fibrin was 1.98 nM, establishing that the PAb has nearly equal affinity for human fgn and fibrin. An avidity of 1x10 10 M -1 /amole lp lipid was found to correspond to the optimal targeting efficiency determined by RIA analysis. We conclude that the ELISA can be employed for routine assessment of EGIL targeting efficiency prior to in vitro and in vivo evaluation. P215 Cyclic AMP-Independent Activation of PKA by Endothelin-1 and Angiotensin II in Vascular Smooth Muscle Cells Nickolai O Dulin, Amanda Davis, Jiaxin Niu, Sergei Orlov. University of Chicago, Chicago, IL; University of Illinois at Chicago, Chicago, IL; University of Montreal Centre de Recherche, CHUM, Montreal, Canada INTRODUCTION. Endothelin-1 (ET1) and angiotensin II (AII) are the vasoactive peptides implicated in cardiovascular pathophysiology. In vascular smooth muscle cells, ET1 and AII stimulate diverse signaling pathways leading to the cell contraction and hypertrophy. RESULTS AND METHODS. Employing an in vivo assay for the activity of the protein kinase A (PKA) in intact cells, we have found that ET1 and AII induce a transient activation of PKA in cultured vascular smooth muscle cells (VSMC) from rat aorta. PKA activity was assessed by phosphorylation of the PKA substrate, vasodilator-stimulated phosphoprotein (VASP), and the specificity of the assay was confirmed by mutation of the PKA phosphorylation site (S153A) of VASP. ET1 and AII did not stimulate cAMP production in VSMC, suggesting a novel cAMP-independent mechanism for PKA activation. The ET1- and AII-induced activation of PKA was found dependent on the inhibitor of kappaB (IB), which is commonly involved in the regulation of the transcription factor NFB. Employing the co-immunoprecipitation technique, we found that a certain pool of IB exists in a complex with the catalytic subunit of PKA, cPKA. Moreover, we demonstrate for the first time that ET1 and AII stimulate a rapid dissociation of the IB/cPKA complex in VSMC. CONCLUSIONS. These data are consistent with the model wherein a distinct pool of IB binds and inhibits the activity of cPKA, whereas the stimulation of VSMC with ET1 or AII results in a release of free (active) cPKA, providing a mechanism for the cAMPindependent activation of PKA. P216 A Heparin-Binding Deficient Mutant of Endothelial Lipase Has Markedly Reduced Catalytic and Lipoprotein Bridging Activities Karen O Badellino, Dawn H Marchadier, Ken Kitajima, Ilia V Fuki, Nadine Blanchard, Jane M Glick, Daniel J Rader. University of Pennsylvania, Philadelphia, PA Interaction with cell-surface heparan sulfate proteoglycans (HSPGs) is important to the catalytic and lipoprotein bridging activities of the triglyceride lipases. The relatively new member of this family, endothelial lipase (EL), contains three conserved putative heparin-binding motifs, two in the 40 kDa amino-terminal portion (Lys 294 to Arg 297 and Lys 306 to Arg 312) and one in the carboxyl terminus (Arg 429 to Lys 434) of the 68 kDa protein. To assess the role of the carboxyl terminal motif, we used site-directed mutagenesis to substitute the 4 basic amino acids with asparagine. A similar mutation in lipoprotein lipase resulted in significantly decreased heparin binding affinity and impaired lipid targeting properties while no changes in the enzymatic activity was observed [Lutz et al, JCI 107:1183–1192(2001)]. Upon transient transfection of 293 cells, the mutant EL protein was produced at similar levels as the wild-type EL protein and exhibited a substantial decrease in heparin binding ability. Interestingly, this heparin binding defective EL mutant had markedly reduced catalytic activity for both triglycerides and phospholipids. Whereas wild-type EL is very effective at bridging LDL to HSPGs on the cell surface, this heparin binding defective EL mutant had virtually no bridging activity. The loss of bridging activity is unlikely to be related to the loss of catalytic activity since heparin-binding competent catalytically inactive EL showed no loss of bridging activity. In summary, the putative carboxyl terminal heparin binding motif in EL is critical for heparin binding, activity, and lipoprotein bridging. Nitric Oxide Inhibits Sustained JNK Activation by Hsp70 Induction in Pulmonary Arterial Hypertension in Rats Shinichi Kanno, Yi-Jen L Wu, Chien Ho, Timothy R Billiar, Larry L Shears II. University of Pittsburgh, Pittsburgh, PA; Carnegie Mellon University, Pittsburgh, PA P217 Background: Pulmonary arterial hypertension (PAH) is associated with dramatic structural changes in the pulmonary vasculature. We have previously shown that nitric oxide (NO) plays a crucial role in this process, however, how NO modulates pulmonary vascular remodeling is not fully elucidated. Heat shock protein 70 (Hsp70), a major stress protein, is induced by NO in many cell types. We postulated that NO modulates vascular remodeling through the induction of Hsp 70 expression in the vessel wall. Methods and Results: Monocrotaline (MCT) was administered to Sprague-Dawley (SD) rats to induce PAH with or without enalapril (ACEI) treatment. Isolated pulmonary perfusion showed ACEI treatment significantly upregulated NO production as measured by nitrite levels. MCT treatment resulted in increased c-Jun N-terminal kinase1/2 (JNK1/2) activation even 5 weeks Downloaded after the treatment. from The addition of ACEI http://atvb.ahajournals.org/ suppressed JNK1/2 activation in association with increased expression of Hsp 70. Coimmunoprecipitation experiments using lysates from the pulmonary vessels revealed that Hsp70 interacts with JNK1/2. Immunohistochemistry demonstrated sustained activation of JNK1/2 in vascular smooth muscle cells (VSMCs) in MCT-treated lungs, whereas Hsp70 was observed in VSMCs in ACEI-treated lungs. Experiments using isolated perfused lungs demonstrated that ACEI treatment was associated with increased NO production while the inclusion of an NO synthase inhibitor induced JNK1/2 activation in the pulmonary vasculature. The capacity of NO to induce Hsp 70 was confirmed using cultured VSMCs exposed to an NO donor or transduced with using an adenoviral vector expressing iNOS. Conclusions: These observations suggest that the mechanism by which ACEI suppresses the development of PAH involves the suppression of JNK1/2 activation with upregulation of Hsp70 induced by nitric oxide. Hsp70 may physically interact with JNK, inhibiting its activation. P218 Artery Calcification and Calciphylaxis are Both Inhibited by Doses of the Amino Bisphosphonate Ibandronate that Inhibit Bone Resorption Paul A Price, Nika Omid, Truclinh Nguyen, Matthew K Williamson. University of California, San Diego, La Jolla, CA The present experiments were carried out to test the hypothesis that there is a common underlying biochemical mechanism that accounts for the different kinds of soft tissue calcification observed in animals that are treated with toxic doses of vitamin D. In previous studies we showed that lethal doses of vitamin D cause extensive calcification of arteries, lungs, kidneys, and cartilage, and that doses of the amino bisphosphonate ibandronate that inhibit bone resorption completely inhibit each of these soft tissue calcifications and prevent death [ATVB(2001)21:817–824]. In the present experiments we have examined the effect of ibandronate on an entirely different type of calcification, the calciphylaxis induced by administration of a challenger to rats previously treated with sub-lethal doses of vitamin D. These studies show that ibandronate doses that inhibit bone resorption completely prevent artery calcification as well as, in the same rat, the Alizarin red-staining for calcification at sites of calciphylactic challenge by either subcutaneous injection of 300g FeCl 3 or intrascapular epilation. Chemical analysis further showed that the level of calcium was increased by 40-fold at the FeCl 3 -induced calciphylaxis site and by 100-fold at the skin epilation-induced calciphylaxis site, and that concurrent treatment with ibandronate reduced calcium to control levels at both sites. Since vitamin D treatment dramatically increased levels of bone resorption, and concurrent treatment with ibandronate normalized resorption, these results support the hypothesis that all soft tissue calcifications in the vitamin D treated rat may be linked to bone resorption. The ability of ibandronate to inhibit vitamin D-associated calcifications in the rat cannot be explained by an effect of ibandronate on serum calcium, since serum calcium remained 50% above control levels in the vitamin D-treated animals that also received ibandronate. Coronary Artery Lipid Accumulation in Diabetic Dyslipidemic Pigs Compared to Normoglycemic Hyperlipidemic Pigs Elzbieta Wysocka, Michael Sturek, Joseph L Dixon. University of Missouri, Columbia, MO P219 We tested the hypothesis that coronary arteries of diabetic pigs fed a high fat (21%)/cholesterol (2%) diet (HFC) would accumulate higher amounts of cholesteryl ester (CE), free cholesterol (FC), and triglyceride (TG) during atherosclerosis than non-diabetic pigs fed the HFC diet. Experimental Design: Five groups of Yucatan miniature pigs (n4–9 pigs/group) were treated for 20 weeks: low fat fed control (C), pigs fed HFC diet (F), low fat fed with alloxan induced diabetes (D), D fed HFC (DF), and mildly diabetic fed HFC (MDF). Methods: Coronary atherosclerosis defined as percent of artery area with raised endothelium was evaluated by intravascular ultrasound (IVUS). Left antherior descending coronary artery (LAD) CE, FC, and TG were measured by HPLC. ANOVA, Student-t, and Mann-Whitney tests were used for statistical analysis. Results: LAD CE, FC, and TG in the unbranched segments were 0.178/-0.043, 33.32/-3.76, and 331.3/-134.7 ug/mg total tissue protein in C pigs. LAD CE concentration increased in F, DF, and MDF 295 (52.6/0.178), 71, and 201-fold, respectively, compared to C (all p0.01). Average LAD FC concentration only increased in F (2.3-fold compared to C, p0.01). Only small increases in LAD TG were observed in HFC fed pigs. Lipid profiles were similar in unbranched and branched LAD segments. Although LAD CE was much greater in F versus DF, IVUS indicated similar sized lesions. Conclusions: 1. CE accumulation in LAD was more accentuated in non-diabetic HFC pigs compared to diabetic HFC pigs. 2. LAD TG was not a marker for HFC or diabetes. 3. Non-diabetic HFC fed pigs develop more CE engorged coronary artery lesions whereas lesions in diabetic pigs may be more proliferative, i.e. cellul Utility of Abnormal 3-Methylglutaconic Aciduria (3MGA) in Diagnosing Statin-Associated Myopathy Paul S Phillips, Richard H Haas, Bruce Barshop, Sergei Bannykh, Darius Amjadi. Scripps Mercy Hospital, San Diego, CA; University of California, San Diego, San Diego, CA P220 HMG CoA Reductase inhibitors (statins) are effective in reducing the incidence of cardiovascular events with a very low rate of toxicity. Generally, patients receiving statins who have muscle symptoms and a normal creatine kinase level (CK) are believed to have symptoms unrelated to this therapy. Yet muscle biopsies confirm myopathy in some patients with normal CPK on statins. We compared the incidence of urine 3-methylglutaconic acid (3MGA) elevation in patients with normal CK and biopsy-proven statin associated myopathy with the incidence in patients with muscle symptoms whose muscle biopsies were normal and with patients on statins without symptoms of myopathy in order to assess the usefulness of this test in diagnosing statin associated myopathy. Methods: We measured 3MGA by standard mass spectroscopy techniques in 8 consecutive patients with biopsy-proven statin associated myopathy, by in 3guest consecutive on April patients 4, with 2013 muscle complaints on statins and negative biopsies,
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