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into fibrin gels by day 7, characterized by dense (242 /- 6 vessels/mm2) networks of small vessels (diameter 10.7 /- 0.4 microns) expressing high levels of VEGF, TGF-b1, bFGF, and Ang-1 throughout the tissue. By 14 days the networks are more dense (326 /- 28 vessels/mm2) with a broader range of vessel sizes (10.7 /- 1.4 microns). VEGF and TGF-b1 levels remain high, while bFGF, and Ang-2 are decreased. Spatially, 14 day TGF-b1 expression localizes to larger vessels in the fibrovascular tissue near the muscle interface. Ang-2 and bFGF expression decreased at the interface but remained high deeper in the gel. ADAM-TS and Plasmin-Mediated Degradation of Versican during Flow-Induced Intimal Atrophy in Baboon Polytetrafluorethylene (PTFE) Grafts P256 Richard D Kenagy, Jens Fischer, Mark Davies, Scott Berceli, John Sandy, Suzanne Hawkins, Thomas Wight, Alexander Clowes. University of Washington, Seattle, WA; University of South Florida, Shriners Hospital, Tampa, FL; Hope Heart Institute, Seattle, WA In a bilateral aorto-iliac PTFE graft model of intimal atrophy in baboons, high blood flow caused by placement of a downstream arterio-venous fistula causes intimal atrophy in the upstream graft. We have investigated whether matrix degrading proteinases are altered in this model of atrophy using the normal flow side (no fistula) as a control. After four days of high flow intimal urokinase was increased (5.03.1 fold of normal flow; p.05; n5) and plasminogen activator inhibitor-1 was decreased (0.430.15 fold of normal; p.05; n5). After 7 days plasminogen, proMMP2, and proMMP9 were increased 3.31.2, 1.230.06, and 1.440.18 fold of normal, respectively (p.05; n5). Extracts of 4 day high flow intimas degraded more 35 S-methionine labeled versican (the major proteoglycan of the intimal matrix) compared to normal flow intimal extracts (114% vs 328% of versican remaining, respectively; p.05; n5). Degradation was inhibited by the serine proteinase inhibitor, AEBSF, and a blocking antibody to plasmin , but not by the MMP inhibitor, BB94. Increased amounts of a 70 kD N-terminal fragment of versican V1 cleaved at E 441 -A 442 were detected in the intima by western analysis (7.24.9 fold of normal flow; p.03; n6) and immunostaining using a neoepitope specific antibody (Sandy et al J Biol Chem 276:13372, 2001). This neoepitope is specific for ADAM-TS enzymes. ADAM-TS4 and ADAM-TS5, but not ADAM-TS1 (TS4 and TS1 are known to cleave versican at E 441 -A 442 ), protein were detected in intimal extracts. While ADAM-TS4 content was not markedly changed, ADAM-TS5 was decreased at 4 days by high flow suggesting other ADAM-TSs mediate 70 kD DPE production. It is also possible that activated ADAM-TSs lose C-terminal TS domains, which mediate binding to the ECM, and are lost from the tissue. In summary, these data suggest that plasmin and ADAM-TS enzymes mediate versican removal during flow-induced intimal atrophy in the baboon PTFE graft. P257 Rho Regulates Extracellular Matrix-Mediated Activation of Arterial Smooth Muscle Cells Joy Roy, Bimma Henderson, Johan Thyberg, Ulf Hedin. Karolinska Hospital, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden The activation of vascular smooth muscle cells (SMCs) from a contractile to a synthetic phenotype is a prerequisite for the migration and proliferation of these cells after arterial injury. This process is dependent on changes in the extracellular matrix composition and includes major changes in SMC structure and function. However, the molecular mechanisms involved are not known. We have previously shown that integrin-linked tyrosine kinase activity and ERK1/2 activation are necessary for phenotypic modulation. The rho GTPases have been shown to regulate signaling pathways that mediate cytoskeletal reorganization and focal adhesion formation. 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to block rho activation by inhibiting the synthesis of mevalonate and its derivatives that are involved in protein prenylation. In this study, we investigated the effect of mevinolin and simvastatin, two lipophilic statins, and the specific rho activation inhibitor, C3 exoenzyme, on phenotypic modulation of SMCs during primary culture of freshly isolated rat aortic SMCs on a substrate of fibronectin under serum-free conditions. Treatment with C3 exoenzyme or statins blocked focal adhesion formation, cell spreading, induction of cyclin D1, and phenotypic modulation as judged by electron microscopy and by western blotting of SMC -actin. Addition of mevalonate and geranyl-geranyl pyrophosphate but not cholesterol or farnesyl pyrophosphate could reverse the effects of mevinolin. In addition, we detected that a smaller fraction of rho was translocated to the cell membrane in statin-treated cells as compared to control cells. These results supported the idea that the geranyl-geranylated proteins such as rho, rac and cdc42 were involved. Our results suggest that rho activation is necessary for SMC phenotypic modulation and that statins inhibit this process by preventing prenylation of rho proteins. Does Manganese Deficiency Reduce Arginase Activity to an Extent Whereby Vascular Function is Altered? Fatima A Tenorio, Jodi L Ensunsa, Carl L Keen, J D Symons. University of California Davis, Davis, CA P258 Arginase is a manganese-containing enzyme that catalyzes the hydrolysis of arginine to form ornithine and urea. Nitric oxide synthase is an enzyme that catalyzes the hydrolysis of arginine to form nitric oxide and citrulline. Animals lacking dietary manganese have reduced arginase activity. We tested the hypothesis that manganese deficiency decreases arginase activity to an extent whereby arginine is diverted through the nitric oxide synthase pathway, resulting in enhanced endothelium-dependent relaxation. Female rats consumed a manganese (Mn) sufficient (45 mg Mn / kg of diet; n6) or Mn deficient diet (0.5 mg Mn / kg of diet, n6) for 632 days. After anesthetizing each animal, sections of liver, abdominal aorta, and the entire heart were excised. Liver Mn (3.40.8 Downloaded vs 41.24.4 nmol/g) from and arginase activity http://atvb.ahajournals.org/ Poster Presentations a-45 (0.70.2 vs 1.90.4 U/mg) were lower (p0.05) in Mn-deficient vs Mn-sufficient animals, respectively. Aortic segments (640 m, internal diameter) and coronary microvessels (118 m, i.d.) were mounted on wire myographs and endothelium-dependent and independent functions were evaluated using acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. In aortic segments that were precontracted with norepinephrine, maximal ACh-evoked relaxation was greater (p0.05) in Mn-deficient (797%) vs Mn-sufficient (549%) animals, while SNP-evoked relaxation was similar between groups (1012% vs 1083%, respectively). In coronary vessels precontracted using endothelin-1, maximal ACh-evoked relaxation (5516% vs 529%) and SNP-evoked relaxation (10521% vs 10310%) were similar in Mn-deficient vs Mn-sufficient rats, respectively. These data indicate that manganese deficiency enhances endothelium-dependent relaxation in aortic but not coronary vessels. Therefore, the vascular effects of reduced arginase activity resulting from manganese deficiency appear to be heterogeneous throughout the vasculature. Role of the Plasminogen System in the Inflammatory Response to Biomaterial Implants P259 Steven J Busuttil, Victoria A Ploplis, Edward F Plow. Case Western Reserve University Cleveland VAMC, Cleveland, OH; W M Keck Center for Transgene Research, University of Notre Dame, Notre Dame, IL; Joseph J Jacobs Center for Thrombosis and Vascular Biology and the Cleveland Clinic Foundation, Cleveland, OH Recent evidence speculates that the plasminogen (Plg) system may play a profound role in mediating cellular recruitment during the inflammatory response. Utilizing a peritoneal biomaterial implant model in mice deficient in components of the plasminogen system, we found that both neutrophil (Neut) and macrophage (M) recruitment was significantly attenuated in the absence of Plg. The present studies were undertaken to elucidate the molecular mechanism by which the Plg system influences the inflammatory response to biomaterial implants. This model involves surgical placement of polyethylene terephthalate disks into the peritoneum of mice. Leukocytes are recovered from peritoneal lavage after 18 hrs and M and Neut populations are distinguished by enzyme activity assays. In mice treated parenterally with tranexamic acid, an antagonist of the lysine binding sites of Plg, there was a significant decrease in both M and Neut recruitment (p0.001). Mice treated subcutaneously with aprotinin, an active site inhibitor of plasmin activity, showed no attenuation in leukocyte recruitment. In addition, mice treated with intraperitoneal aprotinin showed no significant change in M recruitment and only a non-significant decrease in Neut recruitment, although the doses of aprotinin were sufficient to suppress plasmin activity. M and Neut recovered from the peritoneal lavage were used in an in-vitro fibrin degradation assay. M from both wild-type and plasminogen-deficient mice displayed a low baseline ability to degrade fibrin (8.9% and 9.2%, respectively). The fibrinolytic activity of the M increased in the cells of both strains of mice (24.9% and 19.2%, respectively) after the addition of exogenous plasminogen, indicating a similarity in the capacity of the cells to form active plasmin. In conclusion, these studies provide in-vivo verification that the plasminogen system plays an integral role in inflammatory cell recruitment and indicates that the lysine binding sites of plasminogen are critical in this response. Manipulation of the function of the lysine binding sites of plasminogen may provide a means for controlling the inflammatory response to biomaterials. P260 A Dominant-Negative Variant of Nuclear Receptor TR3 Aggravates Vascular Lesion Formation Karin E Arkenbout, Vivian De Waard, Maaike Van Bragt, Tanja A Van Achterberg, Bruno Pichon, Hans Pannekoek, Carlie J De Vries. Academic Medical Center, Amsterdam, Netherlands; IRBHHN, Gosselies, Belgium Vascular smooth muscle cells (SMCs) can undergo relatively rapid and reversible changes in phenotype in response to local environmental alterations, which occur during atherogenesis. Transcription factors are key regulators in phenotypic changes of SMCs. Upon activation of human SMCs with an atherogenic stimulus we found that the mRNA expression of all three members of the Nerve Growth Factor Induced gene-B (NGFI-B) subfamily of nuclear hormone receptors is induced rapidly and transiently. This subfamily comprises TR3 orphan receptor (TR3), Nuclear receptor Of T-cells (NOT) and Mitogen Induced Nuclear Orphan Receptor (MINOR). In addition, NOT is expressed in differentiating THP-1 cells, whereas MINOR mRNA is induced both in THP-1 and MonoMac6 cells. In agreement with the expression profiles of these genes in vitro, TR3, NOT and MINOR mRNA is absent in normal vascular tissue, whereas each of these genes is expressed in neointimal SMCs in human atherosclerotic lesions derived from thirteen different individuals. Especially NOT and MINOR are also expressed in lesion macrophages. To reveal the function of these nuclear receptors in atherogenesis, we applied a variant of TR3 that lacks the transactivation domain (TA) but exhibits normal DNA binding and consequently functions as a dominant-negative inhibitor of all three subfamily members. Adenoviral overexpression of TA in primary SMCs resulted in enhanced DNA synthesis as illustrated by a 10-fold increase in 3H-thymidine incorporation. Homozygous, transgenic mice were generated overexpressing TA under control of the SM22-promoter, resulting in arterial SMC-specific expression. Three founders were bred and show no obvious phenotype and aorta and carotid arteries exhibit normal morphology. Challenge of TA transgenic mice in the carotid artery ligation model results in a 4-fold increase in neointima/media ratio. Based on these data we hypothesize that NGFI-B subfamily members inhibit SMC proliferation in atherogenesis and that the ligands of these orphan receptors may serve as powerful therapeuticby tools guest in vascular on April disease 4, involving 2013 excessive SMC proliferation.
a-46 Arterioscler Thromb Vasc Biol. Vol 22, No 5 Cleaved Fibrinogen ’ Carboxyl Terminus Has Heparin-Like Activity Rehana S Lovely, Lynn K Boshkov, David H Farrell. Oregon Health and Science University, Portland, OR P261 Fibrinogen contains three polypeptide chains, , , and , arranged as a dimer, (, , ) 2. Approximately 10% of fibrinogen molecules contain a highly anionic 20 amino acid sequence in one chain, termed ’, substituted for the carboxyl terminal four amino acids in the more common A chain. Previous studies have shown that the ’ chain binds thrombin, and that the carboxyl terminal 18 amino acids of the ’ chain are cleaved by plasmin during fibrinolysis. Using fluorescence anisotropy, we found that peptides corresponding to the ’ carboxyl terminus bound to thrombin anion-binding exosite II. Exosite II ligands, including heparin, a DNA aptamer directed against exosite II, and a monoclonal antibody directed against exosite II, all inhibited binding of the ’ peptide. In contrast, two hirudin peptides, known exosite I ligands, failed to displace the ’ peptide. We next investigated the enzymatic effects on thrombin of the cleaved 18 amino acid ’ carboxyl terminus. The free ’ peptide had no direct effect on thrombin’s amidolytic activity towards a peptidyl substrate. However, the ’ peptide accelerated the inhibition of thrombin by antithrombin III and heparin cofactor II in a dose-dependent manner. In whole plasma, 500 M concentrations of the ’ peptide prolonged the activated partial thromboplastin time from 29 seconds to 47 seconds. These results indicate that the cleaved ’ peptide has heparin-like activity. Our current hypothesis based on these findings is that the ’ chain provides a high affinity binding site for thrombin during coagulation, in which the active site remains exposed and can continue to convert more fibrinogen to fibrin. This bound thrombin is resistant to heparin inhibition since exosite II is blocked by the ’ chain. However, when fibrinolysis is activated, plasmin cleaves the ’ carboxyl terminus, removing this high affinity thrombin binding site. In addition, the released ’ 18 amino acid peptide inhibits thrombin in the presence of antithrombin III or heparin cofactor II. The ’ peptide may thus provide crosstalk between coagulation and fibrinolysis to prevent further clotting after fibrinolysis is initiated. Gene Transfer of a Novel Chimeric Natriuretic Peptide Shuchong Pan, Laurel S Kleppe, Cheryl S Mueske, Tyra A Witt, Amir Lerman, Robert D Simari. Mayo Clinic and Foundation, Rochester, MN P262 DNP is a recently described member of the natriuretic peptide family produced by the green mamba snake (dendroaspis). Recent studies have suggested unique and potent cardiovascular and renal properties of DNP compared with other family members. Although the amino acid sequence of the mature peptide in the snake is known, neither immature forms of the protein nor the gene encoding for this hormone have been cloned in any species. To develop a system to efficiently express DNP in heterologous systems, we employed preferred human codons for the mature snake peptide cloned downstream of the entire pre-pro sequence of the brain natriuretic peptide (pre-pro BDNP) or the leader sequence of BNP (BDNP) without the prohormone sequences. Transfections in multiple cells (3T3, 293, HepG2) with vectors expressing pre-pro BDNP resulted in nonprocessed (18 kD) forms within cell lysates and a mature form (3kD) in conditioned media. Transfections with vectors expressing BDNP resulted in mature forms within both cell lysates and conditioned media. Functional studies demonstrated the ability of both forms of BDNP to stimulate cGMP production in HUVECs in an endocrine manner. In addition, both forms completely inhibited serum-stimulated (10% FCS) VSMC proliferation. This stimulation of cGMP and inhibition of proliferation in vascular cells is greater than that seen with BNP expressed in a similar manner. When exposed to normal porcine arterial rings BDNP caused significant vasorelaxation (70%) as compared to control media. Intravenous infusion of an adenoviral vector expressing pre-pro BDNP in normal mice resulted in systemic expression of BDNP of 40,000 –70,000 pg/ml for up to 3 weeks compared with undetectable levels in mice infused with a control virus (Ad-LacZ). Expression of BDNP was associated with a decrease in systemic blood pressure as compared to control virus infection. Conclusion: Expression of pre-pro BDNP results in a processed, mature form of BDNP that is able to stimulate cGMP in vascular cells and has potent antiproliferative and vasoactive properties. Adenoviral delivery results in high levels of circulating BDNP in normal mice and decreased systemic blood pressure. P263 Six-Month Patency of ePTFE Grafts Lined with Endothelial Cells Derived from Peripheral Blood Stem Cells Equals Those Harvested from Native Vein Laurace E Townsend, Ahmed A Meguid, John F Knipfer, Phillip J Bendick, John L Glover, Gerald B Zelenock. Wm. Beaumont Hospital Research Institute, Royal Oak, MI Introduction: A prosthetic vascular graft with hemodynamic characteristics suitable for implantation is needed clinically. We compare the outcome of ePTFE grafts seeded with endothelial cells (EC) derived from peripheral blood stem cells (PBEC) versus EC obtained from native jugular vein (JVEC). Methods: Thirteen dogs had EC harvested from excised jugular veins. Stem cells were obtained from peripheral blood by phlebotomy followed by gradient centrifugation. EC developed from stem cells in selective culture. Two 4-mm ePTFE grafts were prepared for each dog; one graft seeded with autologous JVEC and the other with autologous PBEC. The grafts were explanted at 6 mo. and evaluated for thrombus free surface area (TFSA) and for anastomotic intimal hyperplasia. Results: Seven of thirteen animals (54%) maintained at least one patent graft to the six-month end-point. Of fourteen grafts explanted from these animals, 64.3% (9 of 14) were patent; 55.6% (5 of 9) PBEC lined, and 44.4% (4 of 9) JVEC lined, p1.00 by Fishers exact test. There were no significant differences in seeded cell retention, pre-implant confluence, or TFSA. Samples taken from the center of patent grafts stained positively for markers of EC. Comparison of proximal versus distal vessel lumen diameter showed no significant difference (p0.66, exact inference test) indicating that intimal hyperplasia was not a contributing factor to vessel occlusion. Disparity between graft and carotid artery lumen diameter contributed to occlusion (p0.05); graft and native vessel were approximately the same size in 13 of 18 patent anastomoses (72%) and the graft was larger than native vessel at only 1 patent anastomosis. All occluded graft lumens (9 of 9) were larger than native vessel lumen. Conclusions: Considering the highly coagulable canine model used in our study, excellent graft patency results were achieved. Closely matching graft size to vessel size contributes to patency and is a consideration in future clinical trials. Endothelial cells derived from stem cells of peripheral blood are as effective in enhancing graft patency as EC derived from autologous veins. Flow-Induced Actin Dynamics in Living Endothelial Cells Brian P Helmke. Univ. of Virginia, Charlottesville, VA P264 Engineering of artificial vascular grafts requires the establishment of a patent endothelium at the blood-graft interface. Although the mechanisms by which endothelial cells adapt to hemodynamic forces remain unclear, maintenance of endothelial function after graft implant may depend on initial mechanochemical signaling events in response to sudden changes in mechanical environment. Recent measurements of shear stress-induced cytoskeletal displacement in living endothelial cells indicate that intracellular deformation is concentrated at discrete locations. This suggests that force is transmitted via the cytoskeleton to molecular complexes such as focal adhesion sites or intercellular junctions that may integrate mechanochemical signals. Actin microfilaments and stress fibers may play an especially important role due to their structural association with these sites. In the present study, endothelial cells expressing GFP-actin were exposed to changes in unidirectional laminar shear stress in a parallel plate flow chamber. High-resolution optical sectioning microscopy acquired simultaneous fluorescence and DIC 3-D time-lapse images. Fluorescence data was analyzed to demonstrate displacement and dynamics of GFP-actin, and DIC images demonstrated movement of cellular structures such as the nucleus, cell boundaries, and intracellular organelles. After onset of shear stress, ruffles containing F-actin were rapidly projected in an upstream direction, and existing microfilaments were often displaced or bent. In both GFP-actin and DIC images, active remodeling of cell edges was induced within minutes after onset of flow. These results indicate that an increase in shear stress initiates a rapid dynamic response in the actin cytoskeleton that is capable of transmitting force to sites where mechanotransduction occurs. P265 Human Smooth Muscle Calponin Protein is Tightly Restricted to Smooth Muscle Cell Lineages in Transgenic Mice Mary A Georger, Joseph M Miano. University of Rochester Medical Center, Rochester, NY Smooth muscle calponin (SM-Calp) is a highly restricted marker for smooth muscle cells (SMC) whose encoded protein carries out many functions in vitro. Its restricted expression to SMC makes SM-Calp an ideal model gene to dissect the transcriptional circuitry governing SMC differentiation. To date, neither the function nor the transcriptional regulation of SM-Calp has been clearly elucidated in vivo. Accordingly, we generated transgenic mice with a bacterial artificial chromosome harboring the human SM-Calp (hSM-Calp) gene in an effort to understand its transcriptional regulation and begin addressing its function in vivo. The purpose of this study was to optimize conditions for appraising, unambiguously, the expression of hSM-Calp protein in both transgenic embryos as well as postnatal tissues. We took advantage of the fact that the SM-Calp antisera (Dako) used was raised to the human antigen and thus would be expected to have a greater affinity for hSM-Calp than the endogenous mouse SM-Calp protein. We found that the hSM-Calp protein is present in the embryonic heart at 9.5 days and persists throughout development up to 17.5 days, consistent with in situ mRNA hybridization studies. All SMC lineages of the developing embryo show clear hSM-Calp protein with little or no staining of the endogenous SM-Calp protein in non-transgenic embryos. In postnatal tissues, coronary arteries, aorta, and various microvascular beds (brain particularly) show exclusive expression of the hSM-Calp transgene in SMC of these tissues. Notably, staining was also observed in pericytes of the choroid plexus as well as mesangial cells of the kidney. Visceral tissues showing SMC-restricted staining of hSM-Calp include bladder, bronchioles of the lung, as well as the SMC investing the alimentary canal. To date, we have not observed any overt phenotype in these mice. These results demonstrate our ability to clearly discriminate hSM-Calp protein from the endogenous protein using a variety of antigen enhancing protocols. The humanized mice we have generated represent powerful tools to unlock the transcriptional regulation and function of SM-Calp. 3-Dimensional Ultrasound of Extracranial Carotid Arteries: Additional Information? Jörg Nossen, Thomas Vierzigmann, Erich Lang. Waldkrankenhaus St. Marien, Erlangen, Germany Downloaded from http://atvb.ahajournals.org/ by guest on April 4, 2013 P266 Background: External vascular ultrasound is an established method for examination of extracranial carotid arteries. Aim of this study was the evaluation of additional information about carotid stenoses by 3-dimensional sonography. Methods: Patients with suspected carotid stenosis underwent B-mode, doppler and color doppler ultrasound of common carotid (CCA), internal carotid (ICA) and external carotid artery (ECA). Vascular sonography was analyzed for localisation and severity of stenosis. Second step was evaluation of carotid arteries and stenoses by 3-dimensional ultrasound (3-scape power doppler, Siemens Elegra). Afterwards all patients underwent selective angiography of carotid arteries, which was considered as gold standard. Results: 63 carotid segments (CCA, ICA, ECA left and right) of 11 patients were analyzed. 2 segments of one patient could not be 3-dimensionally analyzed because of severe calcifications. Conclusions: Concerning luminal stenoses 80% 3-dimensional ultrasound was
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