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P175 Gene Transfer with Vascular Endothelial Growth Factor Improves Regional Blood Flow and Regional Contractility in a Dose-Dependent Manner in a Chronic Ischemic Pig Heart Model Michael K Hsin, Mark H Danton, Richard Ammer, Kathryn Q Flores, Rita G Laurence, Tom Aretz, Lawrence H Cohn, Lishan Aklog. Harvard Medical School, Boston, MA BACKGROUND: Although gene therapy using plasmid encoding Vascular Endothelial Growth Factor (pl-VEGF) has been shown to promote angiogenesis in ischemic myocardium in animal studies, the optimal dose has not been established. We sought to determine whether the functional benefit of pl-VEGF 165 gene transfer is affected by the dose administered. METHOD: 22 pigs (15–20 kg) underwent placement of ameroid constrictors to the proximal circumflex arteries to produce chronic ischemia. 6 weeks later the animals underwent: (1) sham thoracotomy (n7) or direct intramyocardial injection of pl-VEGF 165 to the ischemic zone using (2) 4.5mg/pig (High Dose, HD, n7) or (3) 0.45mg/pig (Low Dose,LD, n8). 8 weeks later, the following were measured: REGIONAL MYOCARDIAL BLOOD FLOW (RMBF) with radioactive microspheres, and REGIONAL CONTRACTILITY assessed by measuring Fractional Area Change (FAC) with epicardial piezoelectric crystal pairs placed in the ischemic zones. Data was taken at rest and under rapid atrial pacing. RESULTS: In the High Dose group, with rapid pacing, mean RMBF in the ischemic zone was 2.7 times higher compared to the sham group, and the FAC was significantly higher than sham. There was no significant difference between the Low Dose group and sham group at rest or paced, or in the High Dose group at rest. CONCLUSION: Plasmid VEGF gene transfer improves regional blood flow and contractility in a dose-dependent manner in chronically ischemic pig myocardium. Fas (CD95) Sensitizes Macrophages to Oxidized-LDL Induced Death Thomas Q Nhan, W C Liles, Alan Chait, Stephen M Schwartz. University of Washington, Seattle, WA P176 In vitro studies of macrophage death in response to oxidized LDL (oxLDL) were undertaken as a model for the formation of the necrotic core of the atherosclerotic plaque. We showed that thioglycollate-elicited mouse peritoneal macrophages avidly incorporate both oxLDL and acetylated LDL (acLDL) to become foam cells. OxLDL-treated macrophages, but not acLDLtreated macrophages, showed nearly 100% death with characteristics consistent with apoptosis, including cell surface phosphatidylserine exposure, intracellular caspase-3 activity, cleavage of caspase-3 substrates, and DNA fragmentation as shown by TUNEL assay. Surprisingly, the p17 cleaved form of caspase-3, the activated molecule normally found in dying cells, was also present in both control and acLDL-treated macrophages. However, caspase-3 activity was only detected in oxLDL-treated macrophages. The amount of the cleaved form of caspase-3 was reduced by in vitro blockade of FasL with antibody (MFL3) and was absent in lpr macrophages, which lack functional Fas (CD95). Moreover, lpr macrophages resisted oxLDL-cytotoxicity. The naturally occurring Fas-FasL induction of caspase-3 cleavage in living macrophages may, therefore, represent an important physiologic mechanism that sensitizes them to the cytotoxicity of oxLDL. The resulting cleavage of caspase-3 in macrophages is necessary but not sufficient for oxLDL-induced macrophage death. These data suggest that macrophages may exist in an undead state, that represents a functional state dependent on caspase activation. This state appears to be necessary for death in response to oxLDL. P177 Intracellular Metal Chelation Inhibits TNF-Induced Expression of Adhesion Molecules and MCP-1 in Human Aortic Endothelial Cells Weijian Zhang, Balz Frei. Linus Pauling Institute, Oregon State University, Corvallis, OR Endothelial activation and monocyte adhesion are initiating steps in atherogenesis thought to be caused, in part, by oxidative stress. Because redox-active transition metal ions, such as iron and copper, can cause generation of reactive oxygen species and initiation and propagation of lipid peroxidation, they may also cause endothelial activation. Therefore, the objective of the study was to investigate the effects of the iron-chelator desferrioxamine (DFO) and the copper-chelator neocuproine (NC) on TNF-induced expression of adhesion molecules and monocyte chemoattractant protein-1 (MCP-1) in human aortic endothelial cells (HAEC). We found that pre-incubation of HAEC for 16 hr with NC (0.1- 0.5 mmol/L) or DFO (0.01- 0.1 mmol/L), but not iron-saturated DFO, dose-dependently inhibited TNF-induced (10 U/ml) protein expression of E-selectin, VCAM-1 and ICAM-1. DFO at a concentration of 0.1 mmol/L inhibited TNF-induced E-selectin, VCAM-1 and ICAM-1 expression by 742.5%, 843.3% and 591.7%, respectively; and 0.5 mmol/L NC inhibited expression of these adhesion molecules by 933.6%, 843.0% and 894.0%, respectively (P0.01 compared with TNF alone, n 3). In contrast, no inhibitory effect was observed when the cells were co-incubated with DFO plus TNF, or treated for only 1 hr with NC or with bathocuproinedisulfonic acid, a membrane-impermeable copper-chelator, before addition of TNF. DFO and NC also inhibited TNF-induced upregulation of mRNA levels of all three adhesion molecules, as well as MCP-1. Addition of cycloheximide completely abolished the inhibitory effect of NC, but not DFO, indicating that new protein synthesis was required for NC to exert its inhibitory effect. Furthermore, pre-incubation with DFO and NC (0.5 Downloaded mmol/L) inhibitedfrom TNF-induced activation http://atvb.ahajournals.org/ Poster Presentations a-31 of the transcription factor NFB by 34% and 45%, respectively. In conclusion, our data indicate that intracellular transition metals play an important role in cytokine-induced endothelial activation. Therefore, intracellular metal chelation might be a novel strategy to prevent and treat atherosclerosis and other inflammatory conditions. P178 Increased Consumption of Polyunsaturated Fat, Vitamin E and Carotenoids Enhances Brachial Arterial Vasoreactiviy in Men with Coronary Artery Disease Oh Yoen Kim, Ji Young Kim, Jong Ho Lee, Yangsoo Jang, Seok Min Kang. College of Human Ecology, Yonsei University, Seoul, Korea; Cardiovascular Genome Center, School of Medicine, Yonsei University, Seoul, Korea Background: This study aimde to determine whether the isocaloric replacement of cooked refined rice with polyunsaturated fatty acid (PUFA)-rich muffin, composed of walnut, seaweed, sesame oil, and wheat flour in lunch meal reduces coronary artery disease (CAD) risk factors, such as endothelial dysfunction and lipid peroxidation in CAD patients. Methods & Results: Sixty-one male patients with CAD were randomly assigned to either a daily PUFA-rich muffin group or a cooked refined rice group for 8 week. When compared to 307g cooked refined rice, 90g PUFA-rich muffin was isocaloric as 450kcal, but it contained different composition of nutrients. 307g cooked refined rice provided 95g carbohydrate, 7.8g protein, 1.5g fat, 0.31g fiber, 0.86mg vitamin E, 9.45g folate, 1.29g saturated fatty acid (SFA), 1.72g monounsaturated fatty acid (MUFA) and 1.84g PUFA. On the other hand, 90g PUFA-rich muffin provided 42g carbohydrate, 6.2g protein, 28.9g fat, 2.6g fiber, 3.29mg vitamin E, 10.79g folate, 138RE vitamin A, 563g -carotene, 8.08g SFA, 7.21g MUFA and 11.99g PUFA. The muffin group showed the reduction in urinary 8-epiprostaglandin F 2 (PGF 2) (57091pg/mg creatinine vs 48177) without altering body weight and energy intake after 8 week. The baseline urinary PGF 2 was inversely related to brachial arterial vasoreactivity, that is, flow mediated dilation (FMD) (r-0.419, P0.017) and nitroglycerin mediated dilation (NMD) (r-0.553, P0.002), respectively. In the muffin group, FMD and NMD were increased from 4.30.7% to 6.30.9 (P0.05) and from 6.00.7% to 10.60.9 (P0.001) respectively after 8 week. Conclusion- :Increased intake of PUFA, vitamin E and carotenoids via the isocaloric replacement of cooked refined rice with PUFA-rich muffin in a meal exhibited significant beneficial effect on brachial arterial vasoreactivity and on lipid peroxidation in CAD patients. This effect is likely to substantially reduce the risk factors for CAD. P179 Identification of a Pro-Inflammatory CD36-Associated Signal Transduction Pathway in Macrophages Kathryn J Moore, Joseph El Khoury, Lea A Medeiros, Andrew D Luster, Mason W Freeman. Massachusetts General Hospital, Boston, MA The class B scavenger receptor CD36 has been implicated in a wide variety of physiological processes, including atherosclerosis, angiogenesis, lipid metabolism, malarial pathogenesis, tissue homeostasis and Alzheimer’s disease. However, despite its identification more than a decade ago, the physiological events regulated by CD36 ligation remain largely unknown. We have now identified a pro-inflammatory CD36 signal transduction pathway that is induced in macrophages. We demonstrate that -amyloid binds to CD36 on macrophages and microglia, inducing the activation of the Src kinase Lyn and p44/42 mitogen-activated protein kinase. These signaling events require CD36 expression, as -amyloid does not induce p44/42 phosphorylation in macrophages derived from CD36-/- mice. Furthermore, using macrophages derived from mice containing targeted mutations in Src kinase family members, we demonstrate that Lyn, but not Fyn, is essential for CD36 activation of p44/42 MAPK. Interruption of this CD36 signaling cascade in macrophages abrogates pro-inflammatory responses to -amyloid including the production of reactive oxygen species and cytokines. CD36-/macrophages stimulated with -amyloid produced approximately 75–80% less TNF- and MCP-1 as compared to similarly treated wild type macrophages. Comparable decreases of TNF- and MCP-1 were also observed through inhibition of p44/42 MAPK using 5 M PD98059 and in Lyn-/- macrophages, suggesting that interruption at any step of this signaling cascade abrogates these inflammatory responses. In summary, we have identified a -amyloid induced signaling cascade involving the sequential activation of CD36-Lyn-p44/42 MAPK, and resulting in the production of macrophage pro-inflammatory molecules. These data suggest that through its ability to bind modified proteins and lipids such as -amyloid and oxidized LDL, CD36 may play a role in chronic inflammatory conditions such as Alzheimer’s disease and atherosclerosis through induction of a pro-inflammatory signaling cascade. Comparison of Thromboelastography and Aggregometry in Monitoring Glycoprotein IIb/IIIa Inhibition Ramin Artang, Gitte W Jurgensen, Niels J Frandsen, Ulrik Abildgaard, Jorn D Nielsen. Gentofte University Hospital, Hellerup, Denmark; Hilleroed Hospital, Hilleroed, Denmark P180 Objectives: In patients undergoing percutaneous coronary interventions (PCI), there is evidence of fewer coronary adverse events if platelets are inhibited more than 80% with Glycoprotein(GP)IIb/IIIa inhibitors. Maintaining an optimal level of inhibition raises the need for a rapid and reliable bedside monitoring technique. Thrombelastography (TEG), is a bedside method for evaluation of hemostasis. The aim of this study was, for the first time, to compare TEG with aggregometry for monitoring platelet inhibition. Methods: After obtaining informed consent, 10 consecutive patients who underwent PCI and received abciximab were included. Blood was drawn at baseline, and at 2, 8, 24, and 48 hours following bolus and infusion of abciximab (0.25mg/kg, 0.125 g/kg/min for 12 hours). All patients received heparin in cath. lab. and continued on aspirin and clopidogrel. Citrated whole blood was analyzed on modified TEG model 5000 by for guest determination on April of maximum 4, 2013amplitude. Platelet rich plasma was analyzed by
a-32 Arterioscler Thromb Vasc Biol. Vol 22, No 5 turbidimetric aggregometer using 20 M ADP. Levels of inhibition at intervals mentioned above with both methods, were correlated using Spearmans correlation coefficient. Findings: See figure. Conclusion: Percent inhibition of platelets determined by TEG is strongly correlated to the inhibition determined by standard aggregometry. TEG could therefore be useful in rapidly monitoring the effect of GPIIb/IIIa inhibitors. This method is far less time and labor consuming. Larger trials are needed for correlating optimal inhibition as determined by TEG to clinical outcome. P181 Association of Risk Factors of Coronary Artery Disease and Clot Strength Ramin Artang, Esther Jensen, Flemming Pedersen, Niels J Frandsen, Jorn D Nielsen. Gentofte University Hospital, Hellerup, Denmark; Hilleroed Hospital, Hilleroed, Denmark Objectives: The clot strength (the elastic shear modulus of clotting blood) is increased in patients with coronary artery disease. Elevated plasma levels of fibrinogen, C-reactive protein (CRP), homocysteine and cholesterol are also associated with increased risk of coronary artery disease. The aim of our study was to investigate the association of plasma concentrations of these risk markers with clot strength. Methods: After obtaining informed consent, 81 subjects were included: 20 healthy volunteers, 23 with stable angina, 19 with unstable angina (UA) and 19 with myocardial infarction (MI). Upon admission, blood was drawn from the UA and MI patients before antithrombotic treatment. Clot strength in dyne/cm was measured on citrated whole blood by thrombelastography. Plasma concentrations of fibrinogen, CRP, homocysteine and total cholesterol were correlated to clot strength using Spearmans correlation coefficient. Findings: A highly significant correlation was observed between fibrinogen concentration and clot strength (r 0.82, p 0.0001). Clot strength was also logarithmically associated with the CRP level (r 0.54, p 0.0001). There was no correlation between cholesterol level and clot strength (r -0.12, p ns), or homocysteine level and clot strength (r 0.1, p ns). Conclusion: Clot strength is directly correlated to the level of plasma fibrinogen, the structural component of the blood clot. The association between clot strength and CRP level follows a logarithmic pattern. The clinical application of the latter finding requires further investigation. Increased plasma levels of homocysteine and total cholesterol have no direct effect on clot strength. P182 Overexpression of RISC Attenuates Smooth Muscle Cell Growth Responses Jiyuan Chen, Joseph M Miano. Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, NY We and others have shown that all-trans retinoic acid (atRA), a natural retinoid, is effective in suppressing smooth muscle cell (SMC) growth in vitro and inhibiting neointimal formation after vascular injury in vivo. The underlying mechanisms are not clear, but likely involve retinoid-mediated changes in gene expression. Accordingly, we used the Suppression Subtractive Hybridization technique to clone a cluster of retinoid-inducible genes from rat aortic SMC, including a new retinoid-response gene we call retinoid-inducible serine carboxypeptidase (RISC). We have published the work on the cloning, chromosomal mapping and expression profile of RISC. To date, we have been unable to document RISC activity in vitro using well known substrates of other serine carboxypeptidases, suggesting that RISC may catalyze the C-terminal cleavage of distinct proteins. To begin understanding the role of RISC in mediating atRA s biological effects, we generated several SMC lines stably expressing rat RISC under control of the CMV promoter. Of 5 stable cell lines confirmed by Northern blotting to overexpress RISC, 3 were selected for subsequent study. Growth curves revealed that RISC overexpression inhibits SMC growth in vitro. To explore RISC function in cellular signal transduction pathways associated with SMC growth, we examined the effects of RISC overexpression on PDGF-induced MAPK. The results demonstrate that constitutively expressed RISC can attenuate phosphorylation of Erk activated by PDGF in PAC1 SMC. These results provide new insight into RISC function and suggest that this novel retinoid-inducible gene participates in retinoid-mediated events associated with the inhibition of SMC growth. P183 Persistence of the Nitric Oxide Pathway and VEGF-Induced Angiogenesis in Apolipoprotein E-Deficient Mice Nancy Fournier, Ana Fortuno, Nicole Villeneuve, Marie Sauvage, Christine Breugnot, Jean-Pierre Iliou, Nathalie Carpentier, Paul M Vanhoutte, Jean-Paul Vilaine. Institut de Recherches Servier, Suresnes, France Endothelium-derived NO plays role in the regulation of angiogenesis whereas hypercholesterolemia impairs NO release. These two parameters were compared in apolipoprotein E deficient (ApoE KO) and control (C57Bl/6J) mice, 35 weeks old and fed a regular chow. The markers of the bioavailability of NO (relaxation to acetylcholine, Downloaded production of cyclic from GMP) in the thoracic aorta were not different in ApoE KO mice, in comparison with control mice, despite severe hypercholesterolemia, the presence of atherosclerotic lesions, upregulation of MCP-1 expression and increased NADPH oxidase activity in the vascular wall. The VEGF-induced angiogenesis in a sponge implant model was also similar in control and ApoE KO mice. To test if an increased capacity of NO production in these mice could explain the preservation of vascular function and angiogenesis, levels of NOS (type III and II) expression were compared in ApoE KO and control mice using Western blot and immunohistochemistry. The immunodetection signal for NOS III had the same intensity in the two groups of mice. Staining for NOS II was only detected in ApoE KO mice and was mainly localized in the lesion areas. The NOS II detected did not modify vascular reactivity as N-iminoethyl-L-lysine, a specific inhibitor of this isoform, had no effect. The preserved vascular bioavailability of NO and the persistence of the angiogenic capability in ApoE KO mice, which contrast with other models of atherosclerosis or vascular dysfunction, could not be explained by an increased expression of NOS III. An increased activity of NOS III or upregulation of enzymes involved in redox status such as superoxide dismutase, catalase or gluthation peroxidase are alternative explanations to the paradoxical results obtained in this model of atherosclerosis. P184 High Flow Induced Arterial Remodeling Is Associated with Early Expression of TGF-1 and MMPs Eiketsu Sho, Tej M Singh, Mien Sho, Chengpei Xu, Chang Shu, Hirotake Masuda, Christopher K Zarins. Stanford University School of Medicine, Stanford, CA; Akita University School of Medicine, Akita, Japan Arterial adaptation to high flow requires endothelial cell (EC) proliferation, EC migration and internal elastic lamina (IEL) degradation. Recently, transforming growth factor beta 1 (TGF-1) and matrix metalloproteinases (MMPs) have been identified as reg ulators of arterial wall extracellular matrix. We developed a model of acute high flow to identify the time course expression of TGF-1 and MMPs along with arterial remodeling. Arterio-venous fistulae (AVF) were created in Japanese male rabbit s between th e left common carotid artery and the external jugular vein. Animals were sacrificed after 1, 2, and 3 days. Left carotid artery segments were harvested for either RT-PCR with appropriate rabbit primers for TGF-1 and MMP-2, 9 or morphologic examination. Differences between groups were analyzed by ANOVE with Bonferroni correction. On e day after fistula creation, flow increased 5-fold and remained elevated at all time points. Morphologically, high flow resulted in early IEL degradation and fragmentation prior to arterial dilatation. Transcription of TGF-1 a nd MMP-2, 9 was increased as early as one day after flow increase and reduced after 3 days prior to arterial enlargement. We conclude that arterial adaptation to increased blood flow requires IEL degradation and fragmentation prior to enlargement. The ea rly expression of TGF-1 and MMP-2, 9 may provide the initial stimulus to initiate arterial remodeling. Early presence of TGF-1 may initiate eventual expression of MMPs to induce the required IEL degradation. P185 Unsaturated Fatty Acids Inhibit Cholesterol Efflux from Macrophages by Increasing Degradation of ATP-Binding Cassette Transporter A1 Yutong Wang, John F Oram. University of Washington, Seattle, WA Abnormal high-density lipoprotein (HDL) metabolism may contribute to the increased atherosclerosis associated with diabetes and insulin resistance. The ATP-binding cassette transporter ABCA1 is the key mediator of cholesterol transport from macrophages to HDL apolipoproteins. Because fatty acids are elevated in diabetes, we examined the effects of fatty acids on ABCA1 activity in cultured macrophages. Results showed that unsaturated fatty acids markedly inhibited ABCA1-mediated cholesterol and phospholipid efflux from macrophages when ABCA1 was induced by a cAMP analog. This was accompanied by a reduction in the membrane content of ABCA1 and a decrease in apoA-I binding to whole cells and to ABCA1. In contrast, saturated fatty acids were ineffective in these processes. However, when 22(R)hydroxycholesterol was used to induce ABCA1, saturated fatty acids palmitate and stearate inhibited ABCA1-mediated cholesterol efflux, suggesting fatty acid desaturation was also stimulated. Fatty acids did not alter ABCA1 mRNA abundance or incorporation of methionine into ABCA1, indicating that decreased ABCA1 transcription, enhanced mRNA decay, or impaired translation efficiency did not account for these inhibitory effects. Unsaturated fatty acids, however, increased ABCA1 turnover when protein synthesis was blocked by cycloheximide. We conclude that unsaturated fatty acids reduce the macrophage ABCA1 content by enhancing its degradation rate. These findings raise the possibility that an increased supply of unsaturated fatty acids in the artery wall promotes atherogenesis by impairing the ABCA1 cholesterol secretory pathway in macrophages. P186 Hypoxia/Reoxygenation Promotes Myocardial Angiogenesis via NFkB in a Rat Model of Chronic Myocardial Infarction Nilanjana Maulik, Dipak K Das, Shoji Fukuda. University of Connecticut Medical Center, Farmington, CT We examined a novel method of stimulating myocardial angiogenesis by hypoxic precondi- http://atvb.ahajournals.org/ tioning at by bothguest capillaryon andApril arteriolar 4, 2013 levels, and the potential role of NFkB in mediating such
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