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as precise as traditional color doppler ultrasound. In case of severe calcifications 3-dimensional ultrasound may be impossible. Endogenous Smooth Muscle Cell PAI-1: Role in Flow-Induced Smooth Muscle Cell Migration and Regulation of MMP-2 Activity John P Cullen, Eileen M Redmond, Suzanne M Nicholl, Shariq Sayeed, James V Sitzmann, S S Okada. University of Rochester Medical Center, Rochester, NY P267 Several studies have provided compelling evidence for a role of proteases such as urokinase-type plasminogen activators (uPA) and plasminogen activator inhibitor (PAI-1) in regulating smooth muscle cell (SMC) migration. We have previously shown that endothelial cell (EC) PAI-1 inhibits flow-induced SMC migration. In this study we determined the role of SMC derived PAI-1 in the flow-induced migratory process. Wild type (wt) or PAI-1 knockout SMC were cultured in the absence or presence of murine EC under pulsatile flow conditions in a perfused transcapillary culture system. SMC migration was then assessed using a Transwell migration assay. In the absence, but not in the presence of EC, pulsatile flow significantly increased the migration of wt SMC (237 11%, n17, p0.05) when compared to wt SMC cultured under static conditions. While there was no difference in the migration of PAI-1 -/- SMC compared to wt SMC under static conditions, PAI-1 -/- SMC migration was significantly increased under pulsatile flow conditions as compared to wild type controls (334 22% vs 237 11%, n6, p0.05). This flow-induced migration was significantly attenuated, but not completely inhibited, when PAI-1 -/- SMC were cultured in the presence of EC (147 13%, n6, p0.05). uPA activity in PAI-1 -/- SMC exposed to flow increased 354% (137094 vs 30198) in the absence of EC, and 16% (78086 vs 90693) in the presence of EC. However, MMP-2 activity in these cells increased 391% (125920 vs 25623) in the absence of EC and 283% (124642 vs 32504) in the presence of EC. These results suggest that, in the presence of EC, endogenous SMC PAI-1 is required for complete inhibition of flow-induced migration. Furthermore, gene deletion of SMC PAI-1 causes upregulation of MMP-2 activity under flow conditions which may contribute to the flow-induced PAI-1 -/- SMC migratory response observed in the presence of EC. Modification of the Extracellular Matrix by -Irradiation Increases Adhesion and Decreases Migration of Vascular Smooth Muscle Cells P268 Alexandra Kadl, Johannes Breuss, Sophie Ziegler, Florian Gruber, Yuri Koshelnick, Bernd R Binder. Department of Vascular Biology and Thrombosis Research, Vienna, Austria; Klinische Abteilung für Angiologie, Vienna, Austria The limiting factor of percutaneus transluminal angioplasty (PTA) is restenosis, occurring within 6 months in up to 70% of patients with arterial occlusive disease. Restenosisafter PTA is mainly due to migration of smooth muscle cells (SMC) from the adventitia and media into the intima and local proliferation. Partial prevention of restenosis has been achieved by combination of irradiation and PTA. The mechanism by which irradiation prevents restenosis is only partially known and limitation of proliferation of irradiated cells cannot completely explain the beneficial effects. Besides cells, also the matrix is a target for irradiation during the combined PTA and radiotherapy. It was therefore the aim of this study to analyze possible effects of irradiation of the matrix in the absence of cells on SMCs seeded later on such irradiated matrix, specifically on adhesion and migration of these cells. Tissue culture supports coated with vitronectin were -irradiated with 8 Gray. When human arterial SMCs were seeded onto such treated plates, adhesion was significantly increased and migration was decreased compared to cells seeded onto not irradiated plates. Western blots of these cells revealed that on irradiated matrix, the MAP Kinase Erk1/2 phosphorylation was prolonged (3 hrs) as compared to cells seeded onto not irradiated matrix. Such Erk 1/2 activation was due to increased phosphorylation of the focal adhesion kinase indicating activation of intergins. This is consistent with the fact that the increased adhesion on irraditated matrix was RGD dependent. When the effects of irradiation on vitronectin was analyzed further, it was found that irradiation of the protein resulted in a loss of tryptophanes as seen also by oxidation and in fact all irradiation effects were abolished in the presence of an antioxidant. From these data we conclude that -irradiation results in oxidative modification of matrix proteins and in turn increased adhesion of the cells onto such matrix and prolonged activation of the Erk1/2 pathway. Oxidation of extracellular matrix might also contribute to the inhibition of restenosis after radiotherapy. P269 Measuring Unadulterated Whole Blood Viscosity in Patients with Familial Hypercholesterolemia on Long-Term LDL Apheresis Patrick M Moriarty, Cheryl A Gibson, Kenneth R Kensey, William N Hogenauer. University of Kansas School of Medicine, Kansas City, KS; Rheologics, Inc, Exton, PA Background. Recent literature indicates that whole blood viscosity and its major determinants are associated with cardiovascular events and the early stages of atherosclerosis. Major determinants of whole blood viscosity are red blood cell deformability, hematocrit, red blood cell aggregation and plasma viscosity. We undertook this study to evaluate whole blood viscosity changes associated with LDL apheresis (B. Braun, Melsungen, Germany). Unlike conventional viscometers, we were able to measure unadulterated whole blood over a wide shear rate, using new technology that does not require anticoagulants (Rheologics, Inc.), which can severely influence viscosity results. Method. Downloaded We evaluated whole from blood viscosity over a http://atvb.ahajournals.org/ Poster Presentations a-47 wide shear range, and lipid profiles immediately before and after LDL apheresis among patients who were being treated bi-weekly. Results. We assessed five non-obese patients (4 F; 1 M) mean age 57.8 years, who have cardiovascular disease and severe hypercholesterolemia (LDL 200 mg/dl) resistant to diet and pharmacotherapy. Table 1 displays the results for the pre/post comparison of whole blood viscosity and lipid changes associated with LDL apheresis. Conclusion. LDL apheresis resulted in significant reductions in whole blood viscosity at both low and high shear rates. Because elevated blood viscosity may be an important risk factor in atherogenesis, reductions in shear forces by LDL apheresis therapy may play an essential role in both acute and chronic cardiovascular disease. P270 Effect of Intron 4 on eNOS Transcription: Functional Significance of the 27bp Repeats Jian Wang, Donald J Dudley, Xing Li Wang. Southwest Foundation for Biomedical Research, San Antonio, TX; University of Texas Health Sciences Center at San Antonio, San Antonio, TX Endothelial nitric oxide synthase (eNOS) plays an essential role in vascular NO production. We have shown that smoker who had a rare 4 repeat of the 27bp in eNOS intron 4 had increased CAD risk, and associated with a decreased eNOS mRNA and protein levels from placenta tissues. In the present study, we explored the possible functional significance of the 27bp repeat in eNOS gene in transcription efficiency. We inserted 4 or 527bp repeat fragments at either the promoter or the enhancer region of the pGL3 luciferase reporter gene. The constructs with inserts were then transfected to endothelial cells and assessed for transcription efficiency by luciferase activity. We found that the 27bp fragment had a promoter effect when inserted in the promoter region (24.02.1 and 17.00.6 fold for 5 and 4 repeats respectively), which is more efficient than the eNOS promoter fragment. Cigarette smoke-extract significantly up-regulated the promoter efficiency (2.4 and 1.5 folds for the 5 and 4 repeats, respectively). The 27bp repeats also acted as a transcriptional enhancer when inserted at the 3’ region together with the 1.6kb eNOS promoter fragment at the 5’ of the luciferase gene. Vectors containing 5 repeats at the enhancer region had a 8.22.1 fold increase in transcription efficiency as compared to only 3.40.3 fold for the 4 repeats (P0.05). The enhancerless eNOS promoter alone produced a transcription efficiency about 2.80.2 fold of the basic control pGL3 vector. In a mobility shift assay, we observed binding of the 27bp fragment with transcriptional factor(s) from the crude nuclear protein extract of endothelial cells. Our study provides the first evidence that the 27bp repeat in eNOS intron 4 plays a significant role in transcriptional efficiency. Further elucidation of transcriptional effect of the 27bp repeat on eNOS gene, a possible concerted allele-specific effects with eNOS promoter and factors may influence such effect will have significant implications on regulation and protection of endothelial function, hence vascular disease. P271 Matrix Gla Protein (MGP) Expressed in Calcified Lesions of the Aorta Is Inactive as a Binding Protein for Bone Morphogenetic Protein-2 (BMP-2) David C Sane, Andrew Sweatt, Reidar Wallin. Wake Forest University School of Medicine, Winston-Salem, NC Matrix Gla protein (MGP) has an important role as an inhibitor of arterial calcification. MGP is a vitamin K dependent protein that is synthesized by vascular smooth muscle cells and is highly up-regulated in calcified arterial lesions. The mechanism by which MGP works as a calcification inhibitor has been disclosed by our laboratory. MGP is a binding protein for bone morphogenetic protein -2 (BMP-2) and possibly other members of the TGF- superfamily of growth factors. Ligand blotting and affinity chromatography experiments have provided conclusive evidence that the vitamin K modified, -carboxyglutamic acid residues (Gla residues) in MGP are essential for binding of BMP-2. We synthesized a peptide covering the Gla region of MGP and another peptide covering the same region but with Glu residues substituted for Gla residues. Circular dichroism spectra of the Gla peptide in the presence of Ca or EDTA showed that the Gla peptide underwent a Ca - dependent conformational change similar to that demonstrated for the F1 fragment of prothrombin. Both peptides were used to prepare antibodies. The Gla peptide produced conformational specific (“anti-Gla”) antibodies that only recognized the Ca induced conformer of mature and functional MGP. The Glu-peptide produced antibodies (“anti-Glu”), that recognized only immature MGP in which the Glu residues had not been converted to Gla residues. The antibodies were used to investigate MGP in aortas containing calcified lesions. MGP was found to be highly up-regulated in calcified regions of the plaque, but was expressed in a nonfunctional state, since it was detected with anti-Glu but not with anti-Gla antibodies. In contrast, MGP in cartilage was detected exclusively with anti-Gla. The production of Gla-less MGP reveals that there is a functional vitamin K deficiency or a defect in the -carboxylation system in the arterial wall. In conclusion, MGP in calcified arterial lesions lacks the Gla modification and is therefore a nonfunctional inhibitor, being unable to bind to orby inhibit guest the initiation on April of calcification 4, 2013 by BMP-2.
a-48 Arterioscler Thromb Vasc Biol. Vol 22, No 5 Significant Improvement in the Positive Predictive Value of Nuclear Exercise Stress Test by the Presence of Proteinuria P272 Nour M Juratli, Sambit Mondal, Chizor Iwuchukwu, Pejman Shamekh, Sham Maghout, Soad Bekheit, Sheldon Breitbart, Louis Salciccioli, Luther T Clark. SUNY-Downstate Medical Center and Brooklyn Hospital Center, Brooklyn, NY Background: Nuclear exercise stress test (NES) has suboptimal positive predictive value for the presence of coronary artery disease (CAD). Proteinuria is an emerging risk factor for CAD especially in diabetic patients. We intended to study the presence of proteinuria prior to NES to improve its predictive value. Methods: Between 1/99 and 12/00 all patients (pts) who had NES followed by cardiac catheterization (CC) were included in the analysis if they had urinalysis prior to NES. Exercise test database, CC reports, urine chemistry were studied to determine the presence of CAD and proteinuria. Positive NES was defined by the presence of any defect in nuclear images. CAD was defined by the presence of any degree of stenosis during CC. Significant CAD was defined as 50% stenosis in one or more vessels. Severe CAD was defined as 50% stenosis in left main or 70% stenosis in any other coronary artery. Proteinuria was defined as positive if it was present in routine urinalysis at any time prior to NES. Results: Out of 2362 pts who were screened 284 pts had all 3 tests, after excluding pts with known CAD 192 were eligible for analysis. The mean age was 59.4 11.8 yrs. History of diabetes (DM) was present in 54 (28%) pts, hypertension in 117 (61%) pts, and hyperlipidemia in 89 (46.4%) pts. All pts had positive NES. Proteinuria was positive in 55 (28.7%) pts. In all pts NES positive predictive value (PPV) for any CAD had increased from 47.4% to 65.5% (p0.002) by the presence of proteinuria. The PPV for significant CAD had increased from 36% to 47.3% (p0.016). In pts with DM the PPV of NES had increased from 68.5% to 87% (p0.017) for the presence of any CAD. The PPV of NES with the presence of proteinuria did not increase significantly for the presence of severe CAD in diabetic and non-diabetic pts. Logistic regression analysis showed that the presence of proteinuria is an independent predictor of CAD in DM pts (p0.015). Conclusions: Proteinuria is a powerful tool to improve the positive predictive value of nuclear exercise stress for the presence of coronary artery disease. However, it was not associated with angiographic severity of coronary disease. P273 An MRI Technique for the Quantification of Atherosclerotic Lesions in the ApoE Knockout Mouse Stuart M Grieve, David A Priestman, Juergen E Schneider, Fran M Platt, Raymond Dwek, Stefan Neubauer, Kieran Clarke. Oxford Cardiac Research Group Magnetic Resonance Unit, Oxford University, Oxford, UK; Oxford Glycobiology Institute, Oxford University, Oxford, UK; John Radcliffe Hospital, Oxford University, Oxford, UK; BHF Molecular Cardiology Group, Oxford University, Oxford, UK OBJECTIVES: Apolipoprotein-E (ApoE) knockout mice spontaneously develop atherosclerotic lesions and are widely used as a model for human vascular atherosclerotic disease. Standard methods for quantification of atherosclerotic lesions involve serial sectioning of fixed hearts. Lesion areas are typically determined by analysis of digitised images of these sections. We present an alternative method to image excised hearts using MRI after polymer perfusion. METHODS: Mouse hearts were perfused in situ first with heparanised saline, then with a solution of MICROFIL MV-122 (Flow Tech, Carver, Mass). Hearts were dissected out after polymerisation of the perfusate, then embedded in 1% agarose doped with 2 mM Gd-DTPA. MRI experiments were performed using a Bruker Avance spectrometer at 11.7 T. A 512 x 512 x 256 data matrix was acquired using a 3D frequency selective spin-echo method to give a resolution of 31 x 31 x 62 Ã,Âm. RESULTS: Figure A shows a cross-section of the aortic root extracted from a 3D dataset of the whole heart in an Apo-E mouse. The lesion (marked with arrow) can be clearly identified, both from the lumen of the aorta and also from the endothelial wall. Figure B shows a similar section from a wild-type mouse. The wild-type mouse shows a similarly clear definition of the endothelial wall but without the presence of any atherosclerotic lesions. CONCLUSIONS: These initial results clearly demonstrate the potential of combining MICROFIL perfusion and MRI as a tool for accurate and rapid quantification of atherosclerotic lesions in mouse models of human atherosclerotic disease. P274 Simvastatin Inhibits Chlamydia pneumoniae Induced Nuclear Binding of Egr-1 in Mouse Macrophages Florian Bea, Mirja H Puolakkainen, Monica I Shelley, C C Kuo, Lee Ann Campbell, Michael E Rosenfeld. University of Washington, Seattle, WA Activation and nuclear binding of the transcription factor early growth response-1 (Egr-1) is an important modulator of multiple pro-inflammatory genes that are involved in the pathogenesis of vascular disease. HMG CoA reductase inhibitors have anti-inflammatory and anti-thrombotic effects that are independent of their capacity to lower plasma lipids and may involve inhibition of nuclear binding of factors such as Egr-1. To test this possibility, we have investigated whether infection with Chlamydia pneumoniae, an independent risk factor for cardiovascular disease, results in increased binding of Egr-1Downloaded and if this induction from can be inhibited by http://atvb.ahajournals.org/ simvastatin. RAW 264.7 mouse macrophages were infected with Chlamydia pneumoniae or treated with lipopolysaccharide (LPS). Binding of nuclear proteins to Egr-1 consensus oligonucleotide sequences was evaluated by electrophoretic mobility shift assays. Increased binding of nuclear proteins occurred 1–4 hours after infection with Chlamydia pneumoniae or treatment with LPS. Supershift experiments with an Egr-1 specific antibody confirmed binding of Egr-1 to the Egr-1 binding site. Pretreatment of the cells with simvastatin (1M) for 24 hours inhibited the DNA binding of Egr-1 induced by Chlamydia pneumoniae or LPS. These data suggest that infection with Chlamydia pneumoniae may contribute to the development of atherosclerosis via activation of the binding of pro-inflammatory factors such as Egr-1 and that the anti-atherogenic effects of the statins may in part be due to their anti-inflammatory properties. P275 Endothelial Activation and Myocardial Cell Damage Lead to Transplant Coronary Artery Disease and Cardiac Graft Failure Carlos A Labarrere, David R Nelson. Methodist Research Institute at Clarian Health, Indianapolis, IN; Cleveland Clinic Foundation, Cleveland, OH Background. Arterial endothelial activation and microvascular fibrin with myocardial cell damage are independent risk factors for transplant coronary artery disease (CAD) and graft failure. However, little is known about the temporal association between endothelial activation and myocardial cell damage or how these changes relate to outcome. Methods. We evaluated 105 heart transplant recipients 46.6 2.5 months after transplantation for CAD (3.7 0.2 angiograms/patient). All patients survived at least 1 year and had at least a first-year angiogram. Serum soluble intercellular adhesion molecule-1 (sICAM-1) and cardiac troponin I (TnI) were used as markers of endothelial activation and myocardial cell damage, respectively, and were studied using an enzyme-linked immunosorbent assay. Serial samples obtained during the first 3 months after transplantation were evaluated for sICAM-1 while those obtained between 3 months and 1 year after transplantation were evaluated for cardiac TnI. Results. Patients with low serum sICAM-1 (308 ng/ml) during the first 3 months post-transplant and without detectable TnI during months 3–12 after transplantation had significantly less transplant CAD (p0.001) and graft failure (p0.02) compared to patients with elevated sICAM-1 (308 ng/ml) and detectable TnI (table). Conclusions. These data suggest that early activation of the cardiac allograft microvasculature leads to persistent myocardial cell damage and increased transplant CAD and graft failure. The Effects of Isometric Exercise on Haemostasis in Ischaemic Heart Disease Eileen E Mackie, Anna Beattie, Mandy Dawson, Allison McKenzie, Stewart W Hillis. University of Glasgow, Glasgow, UK; Western Infirmary, Glasgow, UK P276 Strenuous dynamic exercise affects haemostasis and may be a trigger for myocardial infarction.However there is minimal knowledge of the effects of isometric exercise in ischaemic heart disease (IHD). This study aimed to examine the effects of isometric exercise on haemostasis in stable angina. Methods: 11 patients with IHD prior to elective angioplasty and 8 healthy controls undertook sustained isometric contraction of their dominant arm to 50% of their maximal voluntary contraction. Haematological parameters were measured before, after and 2 hours following exercise. Flow cytometry using the CD62(p-selectin) antibody was used to assess platelet activation. Results:Expressed as mean(SEM).Paired and 2-sample t-tests were performed. Platelet count increased post exercise in both groups. CD62 was increased only in patients post exercise (2.129(SEM 0.609),1.299(0.319)p 0.05) and remained elevated at 2hr post exercise (2.187(SEM 0.662),1.299(0.319)p0.096).Prothrombin time(PT) shortened in patients post-exercise (12.636(SEM 0.799),12.273(0.856),p0.038) Fibrinogen levels were not significantly different at baseline between the groups (2.83(SEM 0.6),3.015(0.14) p0.846), however there was a reduction at 2hrs in the control group (2.83(0.6),2.62(0.588)p0.049) not seen in the patient group (2.863(0.13),2.986(0.141) p0.178).Tissue Plasminogen Activator (tPA) was reduced in patients at baseline (0.53(0.34),2.98(0.83),p0.023). tPA increased in both groups with exercise(0.53(0.34),2.27 (1.87) p0.377 and 2.98(0.83),3.113(0.975)p0.699). Baseline WCC was significantly higher in patients than in controls (6.88( 0.22),4.96(0.39) p0.001)and increased only in controls with exercise(4.96(0.207),5.328(0.245)p0.002) CONCLUSION: Isometric exercise may be prothrombotic in patients with stable angina relative to controls with increased platelet activation and stimulation of coagulation. Risk stratification using dynamic exercise is well established however isometric exercise induces haemostatic imbalance and is essentially ignored in exercise recommendations by guest on April and risk4, stratification 2013 in IHD.
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