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P47 Effect of Adenosine A 1 Receptor Agonist R-Phenylisopropyladenosine on Left Ventricular Function and Heart Rate in Rats Guo Jin-Ai, Dai Cheng-Xian, Jing Chen. No. 466 Hospital of People’s Liberation Army, Beijing, China Objective To determine the regulative effects of adenosine A 1 receptor agonist R-phenylisopropyladenosine(R-PIA) on the heart rate(HR) and left ventricular function in rats. Methods Forty male Wistar rats were randomly divided into five groups (n8 ): Group normal saline; Group R-PIA 0.03mg/kg; Group R-PIA 0.04mg/kg; Group R-PIA 0.05mg/kg; Group R-PIA 0.03mg/kg plus DPCPX (an adenosine A 1 receptor antagonist) 0.2mg/kg. After being anesthetized, rats were inserted plastic cannula into left ventricular (LV) cavity through carotid artery ,and the hemodynamics indexes of LV were continuously monitored and processed by Cardio2 pressure signal processing software. At different point before and after subcutaneous administration of the drugs , the parameters of HR , LV-PSP, LV-DP, dp/dt max and RPP were recorded . The data were processed by SPSS 8.0 statistics analysis software . Results 1) A dose-dependent negative chronotropic effect on the heart was produced by R-PIA in anaesthetized rats; The decrease of HR was started from 5 min after R-PIA subcutaneous injection and got the lowest value during 15–30 min after administration of R-PIA . The effect of R-PIA on HR gradually disappeared during 90–120 min . 2) The temporary inhibition of LV function induced by R-PIA went to maximum during 15–30 min and tended to weaken at 60 min after administration of the drug . 3) The RPP, an index reflecting myocardial oxygen requirement, was significantly reduced by R-PIA. Conclusions 1) This study validated again that adenosine A 1 receptor agonist R-PIA could bring on a dose-dependent negative chronotropic effect and negative inotropic effect on the heart of rat. 2) R-PIA’s effect of reducing myocardial oxygen requirement could last for a longer time than its negative inotropic effect, and those could be of much benefit to myocardial protection induced by R-PIA. Increased Levels of LDL Oxidation in Patients with Familial Hypercholesterolemia and in End-Stage Renal Disease Patients on Hemodialysis Lambertus J Van Tits, Jacqueline De Graaf, Sebastian J Bredie, Pierre N Demacker, Paul Holvoet, Anton F Stalenhoef. University Medical Center Nijmegen, Nijmegen, Netherlands; Catholic University of Leuven, Leuven, Belgium Patients with familial hypercholesterolemia (FH) and patients with end-stage renal disease (ESRD) undergoing dialysis suffer from accelerated atherosclerosis. Oxidation of LDL is crucial in atherogenesis. Objectives: In the present study, we determined LDL oxidation level of isolated LDL of 11 male patients with FH, 15 male ESRD patients on hemodialysis, and 15 age-matched male normolipidemic healthy controls. FH patients were without lipid-lowering medication for at least 4 weeks and were reassessed after two-year cholesterol-lowering therapy with statins. Methods: LDL oxidation level was measured by ELISA using monoclonal antibody 4E6 to oxidized LDL (oxLDL) as the capture antibody, and anti-human apoB antibody for detection, and expressed as percentage oxLDL. Findings: In FH patients and in ESRD patients on hemodialysis, plasma LDL oxidation levels were significantly elevated compared to controls (4.9 1.3; 3.7 2.0; 1.7 0.6%, respectively). Within each group of subjects, LDL oxidation level was not associated with history of CVD nor with smoking. Furthermore, in neither group a significant correlation was found between plasma concentration of LDL cholesterol and LDL oxidation level. After cholesterol-lowering therapy, LDL oxidation level in FH patients had not changed significantly and remained elevated compared to controls, despite a reduction of LDL cholesterol by 55% on the average. Also absolute plasma oxLDL concentrations, obtained by multiplying LDL oxidation level with plasma LDL cholesterol concentration, were significantly higher in FH patients before and after cholesterol-lowering therapy and in ESRD patients on hemodialysis than in controls (489 145; 189 122; 100 65; 59 27 moles/L, respectively). Conclusion: In conclusion, we show that both in FH patients and in ESRD patients on hemodialysis, oxidation level of circulating LDL is increased, and that cholesterol-lowering therapy with statins does not normalize elevated LDL oxidation levels in FH patients. Increased LDL oxidation levels might be involved in accelerated atherosclerosis in FH and in ESRD. Interaction of Aggregated LDL with Cultured Human Coronary Artery Endothelial Cells Bin Zhao, Wei Huang, Wei-Yang Zhang, Howard S Kruth. Section of Experimental Atherosclerosis, NHLBI, NIH, Bethesda, MD OBJECTIVE: It is known that macrophages take up aggregated LDL (AgLDL), a form of LDL found in atherosclerotic lesions. The purpose of the present investigation was to determine whether cultured human coronary artery endothelial cells (HCAEC) also interact with AgLDL. RESULTS: Both human monocyte-macrophages and HCAEC showed cell-association of LDL aggregated by treatment with sphingomyelinase. After incubation with 50 ug/ml 125I-AgLDL for 1 day, cell-associated 125I-AgLDL was 394 and 455 ug/mg cell protein for macrophages and HCAEC, respectively. When incubated with 50 ug/ml 125I-LDL, macrophages and HCAEC showed very low levels of cell-associated 125I-LDL, 0.40 and 2.10.3 ug/mg, respectively. Interestingly, the microtubule inhibitor, nocodazole, decreased HCAEC cell-associated 125I- AgLDL by 52%, but did not decrease macrophage cell-associated 125I-AgLDL. Degradation of 125 125 125 I-AgLDL (i.e., medium TCA-soluble I) compared with I-LDL was increased 37-fold (from 20 to745 ug/mg) in macrophages, but wasDownloaded not increased forfrom HCAEC (131 and 111 Poster Presentations P48 P49 http://atvb.ahajournals.org/ Poster Presentations a-9 ug/mg). Thus, macrophages and HCAEC degraded 65% and 22% of the total accumulated 125 I-AgLDL (cell-associated degraded), respectively. In contrast to cell-associated 125 I- AgLDL, nocodazole decreased 125 I-AgLDL degradation in macrophages by 38% (from 745 to 464 ug/mg), but did not affect 125 I-AgLDL degradation in HCAEC. This means that although both macrophages and HCAEC can take up and degrade AgLDL, microtubules functioned differently in this process for each cell type. Examination of HCAEC cultures by phase microscopy showed that AgLDL was bound to the surface of some but not all cells. Some HCAEC strains showed low levels of cell-associated 125 I-AgLDL and these same cell strains showed very few endothelial cells that bound AgLDL. CONCLUSIONS: HCAEC process AgLDL differently than macrophages by showing relatively high levels of 125 I-AgLDL cell-association that was nocodazole-sensitive, but low levels of 125 I-AgLDL degradation, which was nocodazole-resistant. In addition, HCAEC strains show cell to cell and strain to strain heterogeneity in their interaction with AgLDL. Lipoprotein Lipid Loading and Cytokine Response in Macrophages Marie Lindholm, Jenny Persson, Jan Nilsson. Lund University, Malmö, Sweden It is well established that macrophage uptake of modified LDL results in foam cell formation, cytokine signaling and most probably propagation of atherosclerotic plaques. However, massive lipid droplet formation and a foam cell like appearance may also be caused by incubation of macrophages with other lipoproteins. The main objective of the current study has been to study how such lipid loading affect macrophage inflammatory response. Human monocyte-derived macrophages have been pre-incubated with fresh LDL, vortex-aggregated LDL (AgLDL) or VLDL, which increase intracellular cholesterol esters or triglycerides, respectively. Cells were differentiated for five days before commencing the experiment, then pre-treated with respective lipoprotein for 16 h. Cells were incubated for 4 h with fresh media with or without TNF- .Cytokine concentrations have been measured in conditioned media and lipid content in the cells (n10). It appears as if the lipoproteins have differential effects on both basal and stimulated cytokine secretion. Pre-treatment with VLDL increase basal secretion of IL-1 with 230% compared to control, and decrease secretion of TNF- by 57% and IL-8 by 70%. AgLDL have a less pronounced but similar pattern of effects on cytokine secretion (160, 51 and 50%, respectively), as does native LDL (70, 44 and 50%, respectively). The cells were also stimulated with TNF- after pre-incubations with lipoproteins. VLDL-treatment decreased stimulated IL-8 secretion by 32% compared to control, whereas LDL and AgLDL had mild increasing effects. In contrast, AgLDL had a strong enhancing effect on stimulated IL-1 secretion (80% more than control) compared to LDL and VLDL (8 and 40%). In conclusion, it appears as if LDL, AgLDL and in particular VLDL have specific effects on basal and cytokine-stimulated macrophage inflammatory signals in vitro. This is of interest as VLDL clearance is slow in diabetic patients and it has long been recognized that this may be causative to the increased risk for atherosclerosis in these individuals. P51 Overexpression of Antioxidant Enzymes Reduces OxLDL-Induced Monocyte Adhesion to Mouse Endothelial Cells Zhongmao Guo, Hong Yang, Felicia Rymundo, Arlan Richardson. Meharry Medical College, Nashville, TN; University of TX Health Science Center at San Antonio, San Antonio, TX Adhesion of monocytes to endothelium plays a critical role in atherogenesis. In the present study, we determined the effect of overexpressing antioxidant enzymes on the adhesion of monocytes to endothelial cells (EC) and the expression of EC adhesion molecules. ECs were obtained from the aorta of wild-type mice and transgenic mice overexpressing Cu/Znsuperoxide dismutase (SOD), catalase or both Cu/Zn-SOD and catalase. The ECs were incubated with native LDL, CuSO4-oxidized low-density lipoprotein (oxLDL) or culture medium alone (control) for 2 hours and then incubated with mouse monocytes for 1 hour. Adhesion of monocytes to ECs was determined by measuring the myeloperoxidase activity or the number of monocytes firmly attached to ECs. The expression of EC adhesion molecules was determined by measuring the protein level of vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemotactic protein 1 (MCP-1) after the ECs were incubated with native LDL, oxLDL or culture medium alone. Results from this study showed that oxLDL significantly increased monocyte adhesion to ECs and significantly increased the protein level of VCAM-1 and MCP-1 in ECs, overexpression of antioxidant enzymes significantly reduced oxLDL-induced monocyte adhesion to ECs and significantly reduced the level of EC adhesion molecules. For example, after ECs were treated with oxLDL, the number of monocytes attached to the ECs obtained from transgenic mice overexpressing Cu/Zn-SOD, catalase or both Cu/Zn-SOD and catalase was 50%, 30% and 70% less, respectively as compared to that attached to ECs obtained from wild-type mice. The protein level of VCAM-1 and MCP-1 in ECs obtained from mice overexpressing Cu/Zn-SOD and/or catalase was significantly lower than that in ECs obtained from wild-type mice. These results suggest that reactive oxygen species, such as superoxide and hydrogen peroxide, play a role in oxLDL-induced monocyte adhesion to ECs. This work is supportedby in part guest by AHA on grant April 0030239N 4, 2013and NIH grant 1P30AG13319. P50
a-10 Arterioscler Thromb Vasc Biol. Vol 22, No 5 P52 Conformational Activation and Receptor Co-Association of GpIIb/IIIa on Platelets Stefan Barlage, Alexandra Pfeiffer, Antonia Wimmer, Gregor Rothe, Gerd Schmitz. Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany Analysis of the fluorescence resonance energy transfer between pairs of fluorochrome labeled antibodies enables the analysis of receptor conformation or receptor clustering. The alphaIIb/ beta3 integrin (GpIIb/IIIa) is expressed on platelets, where it acts upon conformational activation as a fibrinogen receptor, playing a critical role in platelet aggregation. GpIIb/IIIa antagonists have been accused to cause occasional thrombocytopenia, probably via drug induced platelet activation or immunogenic neoepitopes. Binding of the GpIIb/IIIa receptor antagonist MK-383 induced a GpIIb/IIIa conformation which differed from both the resting and the ADP-activated receptor but there was no effect on P-selectin expression, indicating that MK-383 binding did not induce general platelet activation via activated GpIIb/IIIa receptors. The interaction of GpIIb/IIIa with other surface receptors may further modulate the signaling associated with GpIIb/IIIa activation. Moreover, hypercholesterolemia or hypertriglyceridemia are characterized by an increased in vivo activation of platelets and an altered membrane fluidity as determined by pyrenedecanoic acid monomer/excimer distribution. It is tempting to speculate that functional receptor co-operation, probably within cholesterol/sphingolipid rich membrane domains is a major regulatory principle of platelet activation. We could demonstrate activation dependent changes in the composition of a GPIIb/IIIa associated receptor cluster. GPIIb/IIIa is associated with CD36 ex vivo. Upon activation, however, CD36 is separated from the complex and the Fcgamma-receptor II (CD32) is included into the cluster. Concomitantly, upon activation, CD32 associates to an integrin associated protein (CD9). In summary, the interaction of GpIIb/IIIa with other receptor molecules or integrin associated molecules may represent an important regulatory mechanism of platelet activation. P53 p38 MAP Kinase Regulates Interleukin-1 Synthesis by Dissociating mRNA from TIA-R in Activated Platelets Stephan Lindemann, Neal D Tolley, Thomas M McIntyre, Guy A Zimmerman, Andrew S Weyrich. Human Molecular Biology and Genetics, Salt Lake City, UT We have previously demonstrated that platelets synthesize IL-1 (Interleukin-1) in an activation-dependent manner. Here we delineate the specific signaling pathway that regulates IL-1 synthesis by platelets. Activated platelets synthesize the majority of pro-IL-1 by 8 hours and subsequently process this precursor into its mature form (between 200-1000 pg/ml) in a concentration and agonist-dependent manner. When platelets are pretreated with the p38 MAP-kinase inhibitor SB203580, but not an inactive scrambled derivative, IL-1 synthesis is completely abrogated (below 10 pg/ml). p38-dependent IL-1 synthesis is consistent with a rapid increase in p38 MAP kinase phosphorylation that is sustained over an 8 hour time period. Neutralization of p38 MAP kinase activity also blocks eIF4E (eukaryotic Initiation Factor-4E) phosphorylation. However, it does not prevent eIF4E from binding IL-1 mRNA in activated platelets. In a search for other RNA binding proteins that may regulate the translation of IL-1 mRNA in a p38-dependent fashion, we isolated proteins bound to poly-adenylated mRNAs that were extracted with oligo-dT beads. In these studies, we found that platelet activation increased the number of proteins bound to mRNAs compared to resting cells. In addition, separation of the proteins by 2-dimensional SDS-PAGE demonstrated that pretreatment of the activated platelet suspensions with the p38 MAP kinase inhibitor captured 2 additional RNA-binding proteins. Predictions based on the molecular weight and PI of these proteins indicated that they were from the TIA family of translational repressing RNA-binding proteins. Immunocytochemistry and western analysis confirmed the presence of TIA-R in platelets and subsequent studies demonstrated that TIA-R binding to IL-1 mRNA is markedly increased in activated platelets pretreated with the p38 MAP kinase inhibitor. These studies suggest that the p38 MAP kinase pathway modulates RNA binding to translational repressing TIA proteins. They also suggest that p38 MAP kinase inhibitors may be useful clinical tools for inhibiting cytokine synthesis by aggregated platelets. P54 Are Routine Investigations for Hypercoagulable Disorders Justified in Acute Ischemic Stroke? Majaz Moonis, Timea Kovats. UMass Medical School, Worcester, MA Introduction. Indications for hypercoagulable work-up in acute ischemic stroke are unclear. Under current recommendations hypercoagulable work-up is not recommended if other risk factors present might explain the basis of the stroke. However a hypercoagulable workup is expensive. Under these circumstances, is it useful to screen patients with acute ischemic stroke for hypercoagulable states? Methods. We performed a retrospective cohort study of all consecutive patients admitted with acute ischemic stroke between January, 1999-March, 2001.The objective was to determine the prevalence of hypercoagulable risk factors and their concomitant association with other vascular risk factors. Work-up in all cases included Lupus Anticoagulant, Anticardiolipin antibodies, Antithrombin 111, protein S and C deficiency, Factor V Leiden, Prothrombin 20210A gene mutation and Fibrinogen. Results.Of the 283 patients , hypercoagulable work-up had been done in 53(18.7%)and a positive result was seen in 23(43.4%). Antithrombin 111 ,Protein S and Protein C deficiency were seen in 52%, 17.3% and 21.7% respectively. Lupus Anticoagulant and Anticardiolipin antibodies were detected in 30.4%. Elevated fibrinogen levels were evident in 13%. In patients with abnormal results stroke types were as follows: 52.3%had embolic, 17.3% thrombotic,13.2% lacunar and 17.3% indeterminate. In 32(60.2%) other concomitant risk factors were identified. Diagnosis of hypercoagulable states led to a significant increase in the percentage treated with anticoagulants: heparin (9%) and warfarin (22%). Death in 5 patients was associated with 2 Antithrombin 111 levels. Conclusions. Hypercoagulable Downloaded states appear from to be more common than http://atvb.ahajournals.org/ currently believed and co-exist with other stroke risk factors including underlying cardiac disease.In patients with large vessel non-cardioembolic strokes where anticoagulation is usually not anticipated, evaluation for a hypercoagulable state may effect therapeutic decisions. This may reduce stroke recurrence and other co-morbid conditions such as deep vein thrombosis and pulmonary embolism. Comparative results with studies will be discussed. P55 Functional Properties of the Endothelial and Media Layers of the Human Tissue-Engineered Blood Vessel Murielle Remy-Zolghadri, Guillaume Grenier, Karina Laflamme, Lucie Germain, Francois A Auger. Laval University, Quebec, QC, Canada We have reported the production of a human TEBV by the self-assembly approach as the first step in the development of a small diameter vascular prosthesis. We present herein the characterization of physiological functions of the endothelial and media layers. The role of endothelial cells (EC) in the hemostatic balance and inflammatory process was assessed by quantification of vWF, thrombomodulin (TM) and proadhesive molecules. The function of the reconstructed media (RM) was analyzed by the detection of contractile proteins in SMC, the accumulation of extracellular matrix and their organization in the media. The vasocontractile response of the RM to endothelin was also evaluated. Results show that the endothelium (50 000 5400 EC/cm2) presents a basal secretion of procoagulant vWF that increases twice after stimulation. Eighty percent of EC express the anticoagulant TM at any time of the maturation process. Twenty four hours after seeding, 1.26 3.3 and 2.3 2.2 % of cells express E-selectin and ICAM-I, respectively. As expected, cells up-regulate the expression of these molecules when stimulated. Concerning the RM, results show that the tension is a key factor for SMC survival and maturation since cells in a loose environment dedifferentiate. In contrast, when a tension is applied, SMC become aligned in the axis of the tension and a compaction of the ECM occurs. From a cellular point of view, SMC up-regulate their secretion of collagen IV whereas collagen I is down-regulated. Differentiation markers increase as a function of culture time and ECM remodeling. RM respond to endothelin (EC50 of 5nM) and SMC express Et-A receptors since contraction was diminished by BQ-123 antagonist. In summary, the endothelium displays anticoagulant properties associated with no proinflammatory characteristics. The vascular media contains differentiated SMC aligned in an organized ECM. Moreover, SMC are able to react to endothelin by expressing Et-A receptors. Taken together, these results show that cells in the TEBV possess physiological functions normally present in blood vessels, indicating the potential of these living tissue constructs as vascular prosthesis. P56 WITHDRAWN P57 Cyclooxygenase-2 Is Induced in Monocytes by PPAR and Oxidized Alkyl Phospholipids from Oxidized LDL Thomas M McIntyre, Aaron V Pontsler, Andy St Hilaire, Gopal K Marathe, Guy A Zimmerman. University of Utah, Salt Lake City, UT LDL oxidation and monocyte infiltration of the vessel wall underlie atherogenesis. These cells express cyclooxygenase-2, but the mechanism by which oxidized LDL stimulates cyclooxygenase-2 transcription is unknown. Circulating human monocytes do not contain PPAR, but we find that engagement of their surface ICAM-3 induces PPAR message and protein accumulation within a few hours. Oxidatively-fragmented phospholipids isolated from oxidized LDL, a synthetic oxidized alkyl phospholipid (azPC) that is a potent PPAR agonist, or rosiglitazone induced cyclooxygenase-2 expression and enhanced PGE 2 secretion. Phospholipase A 1 and A 2 digestion demonstrates that oxidized alkyl phospholipids, and not oxidized fatty acids, were the relevant agonists. Cyclooxygenase-2 induction only occurred in monocytes containing PPAR. The cyclooxygenase-2 PPRE was required for induction by oxidized phospholipids, and these lipids, azPC, and rosiglitazone stimulated full length and (Cox-2 PPRE) 3-luciferase reporters. The cyclooxygenase-2 PPRE responded strongly to PPAR agonists, less well to WY14643 at concentrations selective for PPAR, while higher concentrations of 15-deoxyPGJ 2 and ciglitazone were inhibitory. 15-deoxyPGJ 2 inhibited PMA-induced cyclooxygenase-2 expression by PPRE-dependent and -independent mechanisms. Thus PPAR is rapidly induced in primary monocytes by appropriate outside-in signaling. This allows them to respond to previously undetectable agonists in oxidized LDL. Vascular cells such as endothelial cells and smooth muscle cells constitutively express PPARgamma and are not subject to this form of regulation. Cyclooxygenase-2 and PGE 2 secretion are induced - not inhibited - by selective PPAR agonists that includes oxidativelyfragmented phospholipids in oxidized LDL. Ceramide Upregulates Nitric Oxide Synthase Expression in Human Endothelial Cells Huige Li, Peter Junk, Christian Burkhardt, Thomas Wallerath, Ulrich Förstermann, Andrea Huwiler, Josef Pfeilschifter. Johannes Gutenberg University Mainz, Mainz, Germany; Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany Ceramide is a sphingolipid second messenger that can be formed by hydrolysis of membrane sphingomyelin by sphingomyelinase. Recent studies have indicated that ceramide can induce vasodilation and can activate endothelial NO synthase (eNOS) through a Ca2-independent mechanism. The aim of this study was to examine whether ceramide modifies the expression of eNOS gene. Methods: eNOS mRNA and protein expression in human umbilical vein endothelial cells (HUVEC) and in HUVEC-derived EA.hy 926 endothelial ce lls were analyzed by RNase protection assay and Western blot, respectiviely. A 1.6-kb human eNOS promoter fragment by cloned guest before on the April luciferase 4, 2013 gene was transfected transiently into EA.hy 926 P58
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