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P107 Cardiac Ankyrin Repeat Protein Expression in Human and Murine Atherosclerotic Lesions: Activin A Induces CARP in Smooth Muscle Cells Vivian De Waard, Tanja A Van Achterberg, Nicholas J Beauchamp, Hans Pannekoek, Carlie J De Vries. Academic Medical Center, Amsterdam, Netherlands Cardiac ankyrin repeat protein (CARP) is a transcription factor related protein. By using extensive serial analysis of gene expression (SAGE) we previously identified CARP as one of the genes induced under atherosclerotic conditions both in cultured endothelial cells and vascular smooth muscle cells (SMCs). In the present study, we investigate the expression of CARP in human and murine atherosclerotic lesions by in situ hybridization. CARP expression is observed in endothelial cells lining human atherosclerotic plaques, whereas lesion macrophages are devoid of CARP. Furthermore, we establish that CARP mRNA and SM -actin antigen co-localize in a subset of neointimal SMCs, while no CARP mRNA is encountered in medial SMCs. Since the CARP mRNA-expressing neointimal subset of SMCs is distinct from neointimal SMCs that synthesize the activation marker osteopontin, it is deduced that CARP is specifically expressed in (re)differentiating intimal SMCs which become quiescent. Finally, we show that activin A, a member of the TGF superfamily that enhances (re)differentiation of SMCs, induces CARP expression in SMCs. It is argued that our data support the view that CARP is involved in (re)differentiation of SMCs during atherogenesis. P108 Induction of SM22 Alpha Promoter Activity by Vasopressin and Suppression by Platelet-Derived Growth Factor Involve Distinct Regulatory Elements in Vascular Smooth Muscle Cells Nihal Kaplan-Albuquerque, Chrystelle Garat, Vicki Van Putten, Raphael A Nemenoff. University of Colorado Health Sciences Center, Denver, CO Development of VSMC involves the coordinated induction of a number of smooth muscle specific markers. SM22alpha has been used as a marker to study the differentiated state of VSMC. Under certain pathophysiological states, VSMC decrease expression of contractile proteins, and convert to a more proliferative phenotype. Relatively little is known about the pathways controlling regulation of SM22 expression by circulating factors. In this study, control of SM22 expression in adult VSMC was examined. High basal SM22 mRNA levels and promoter activity in cultures of rat VSMC were markedly inhibited by PDGF. Stimulation with AVP increased SM22 mRNA levels and caused a 2–4-fold increase over basal promoter activity, which was blocked by PDGF. SM22 promoter has three CArG elements, one E-box and two GC boxes. Expression of a fragment containing only one CArG box was sufficient to drive AVP stimulated promoter activity. Mutations in near CArG and GC boxes reduced the basal and AVP stimulated promoter activity but had no effect on suppression by PDGF. Similarly, overexpression of SRF enhanced the basal and AVP stimulated activity of fragments with CArG boxes but did not affect PDGF suppression. In contrast, over-expression of Sp1 inhibited the promoter activity. By mutational analysis and EMSAs, the GC box did not appear to be the site for Sp1 binding. However, Sp1 bound to a GC-rich region 3’ to the GC box. Expression of gain of function Ras and Rac1, but not RhoA and Cdc42, potently suppressed SM22 promoter activity. Inhibition of p38 MAP kinase and PI-3 kinase inhibited AVP stimulated promoter activity, whereas, inhibition of ERKs had no effect. None of these agents affected PDGF-induced suppression. These data indicate that in the regulation of SM22 expression, Vasoconstrictors appear to act through a pathway that involves p38 MAP kinase and PI-3 kinase, whereas PDGF suppression may be mediated through Ras and Rac1. These signaling pathways are likely to act through distinct transcription factors, which either lead to induction of promoter activity (SRF) or suppression (Sp1). Shear Stress Induction of p21 waf1/cip1 Rescues Endothelial Cells from Apoptosis During Cobalt Chloride-Simulated Hypoxia Carlo Gaetano, Stefania Mattiussi, Fabio Martelli, Laura M Barlucchi, Annalisa Antonini, Roberta Palumbo, Barbara Illi, Corrado Cirielli, Chiara Nicolò, Roberto Testi, Lucia Testolin, Francesco Osculati, Giulio Pompilio, Maurizio C Capogrossi. Istituto Dermopatico dell’Immacolata, Roma, Italy; Istituto Cardiologico “Fondazione Monzino”, Milano, Italy; Istituto Cardiologico “Fondazione Monzino”, Roma, Italy; Università degli Studi “Tor Vergata”, Roma, Italy; Università degli Studi di Verona, Verona, Italy P109 Hypoxia was simulated by addition of CoCl2 to human umbilical vein endothelial cells (HUVEC) cultured in absence of growth factors. After 12 hours of this treatment (T0), flow cytometry, ELISA and pulse field analyses revealed the initial onset of an apoptotic process. At T0, HUVEC were exposed to laminar shear stress (SS) (15 dyne/cm2 /sec-1 ) for additional 2 to 12 hours or kept in static condition (ST). SS induced cell cycle arrest in G0/G1 preventing accumulation of cells with sub-2N DNA content, chromatin fragmentation and poly(ADP-ribose)polymerase (PARP) cleavage while cells in ST underwent significant DNA degradation. In this experimental setting, targeting p53 tumor suppressor by papilloma E6 protein expression significantly reduced the number of apoptotic cells detected in hypoxic ST culture at any time point. Notably, in wild-type as well as p53 deficient HUVEC, SS progressively increased p21Waf1/Cip1 protein levels reaching a peak between 4 to 6 hours. In order to investigate its functional role, a sense (Sp21) and antisense p21Waf1/Cip1 (Asp21) were expressed in cells kept in ST or in presence of SS by recombinant adenoviruses (AdCMV.Sp21 and AdCMV.ASp21) infection. Remarkably, AdCMV.Sp21 infected HUVEC, cultured in ST, were protected from death to a similar extent as mock infected cells exposed to laminar SS. Conversely, the expression of ASp21 significantly reduced SS protection of HUVEC from apoptosis. These results show that p21Waf1/Cip1 induction, occurring in presence of SS, is required for preventing human endothelial cells to undergo apoptosis during hypoxia . Downloaded from http://atvb.ahajournals.org/ P110 Analysis of the Tissue Factor Pathway Inhibtor Gene and Antigen Levels in Relation to Venous Thrombosis Ali A Nekoo. University of Leeds, Leeds, UK Poster Presentations a-19 TFPI inhibits tissue-factor induced coagulation. The major part of TFPI is releasable by heparin. We recently found eight patients with thrombosis and low levels of heparin-releasable TFPI. The aims were to investigate the TFPI gene for mutations. A transition of G to A coding for Valine264Methionine in the heparin-binding domain was found. The Val264Met polymorphism had an allele frequency of 3% in 96 healthy individuals. A silent polymorphism was identified in TFPI exon IV (TÂ®C), which does not alter Tyrosine 56. Apart from Val264Met which was detected in one out of the eight patients, no other mutations in the TFPI gene were found in patients with low heparin-releasable TFPI. Analysis of Val264Met in 317 patients with DVT and 292 controls showed no association between Val264Met and DVT. However a study of total TFPI antigen levels in 122 DVT patients and 126 controls demonstrated an association between TFPI levels and venous thrombosis (p0.0001). These results provide an evidence for a relation between venous thrombosis and Total TFPI level as a possible risk factor, whereas they do not support a link between DVT and mutations in the nine exons of the TFPI gene. P111 Human Adipose Stromal Cells Express the Angiogenic Factor VEGF and Its Receptor VEGFR-2 Jalees Rehman, Jingling Li, Catharine A Williams, Sebastian Bekkers, Robert V Considine, Keith L March. Indiana University and Indiana Center for Vascular Biology, Indianapolis, IN Background: The delivery of stem and progenitor cells to augment angiogenesis is emerging as a novel therapy. One obstacle is the limited availability of such cells for autologous delivery. The recent discovery that the adipose stromal fraction contains pluripotent stem cells suggests that it may be a source of cells for therapeutic angiogenesis. We examined the ability of cultured adipose stromal cells to secrete the angiogenic factor VEGF (Vascular endothelial growth factor) and express the stem cell marker AC133 as well as VEGF receptor VEGFR-2 (KDR). Methods: Subcutaneous adipose tissue biopsies were obtained from obese patients. The adipose stromal fraction was separated from the adipocyte fraction by centrifugation. Cells were cultured in either DMEM (Dulbecco’s Modified Eagle Media) or EGM2-MV (Endothelial Growth Media-2 MV, Clonetics). To determine VEGF production, cell cultures were switched to media without supplemental growth factors for 48 hrs. Supernatants were subsequently assayed for VEGF by ELISA. The cell surface expression of VEGFR-2 and the stem cell marker AC133 were determined by flow cytometric analysis. Results: The VEGF concentration in the supernatants of adipose stromal cells cultured in DMEM was 526 pg/ml as compared to 279 pg/ml when cultured in endothelial media. The percentage of cells expressing the VEGF receptor KDR was 2.4% in DMEM and 1.45 % in endothelial media. The endothelial media favored the expression of the stem cell marker AC133 with 1.5% positive cells when compared to only 0.6% in the DMEM. Conclusions: These experiments demonstrate for the first time that stromal cells obtained from the easily accessible subcutaneous adipose tissue are able to secrete VEGF and also express the VEGF receptor VEGFR-2. Furthermore, the stem cell marker AC133 can be found in the adipose stromal fraction, thus supporting previous reports that the adipose stromal fraction contains pluripotent stem cells. These findings suggest that subcutaneous adipose stromal cells may be an attractive candidate for autologous cell delivery in patients with athersclerosis to augment angiogenesis. P112 Transcriptional Profile of Human Endothelial Cells Exposed to Fluid Shear Stress Scott M Wasserman, Fuad Mehraban, Laszlo Komuves, James E Tomlinson, Ying Zhang, Frank Spriggs, James N Topper. Stanford University, Stanford, CA; CuraGen Corporation, New Haven, CT; COR Therapeutics, South San Francisco, CA Hemodynamic forces generated by blood flow play an integral role in vascular homeostasis. In vitro biomechanical forces such as fluid shear stresses, modulate endothelial phenotype, in part, through changes in gene expression. We employed the high throughput expression profiling technique GeneCalling® to evaluate the mRNA transcript profile of human umbilical vein endothelial cells exposed to a steady laminar shear stress at 10 dynes/cm2 (an arterial magnitude of shear stress). A total of 35,985 bands, corresponding to cDNA fragments resulting from digests with 93 pairs of restriction enzymes, were detected. Four hundred eighty four bands demonstrated a greater than two fold change in expression (up/down) at 24 hours of laminar shear stress compared with the no flow control in triplicate experiments. Differentially expressed bands were queried against sequence databases, resulting in the identification of 108 distinct genes. Approximately 15% of these represent species whose response to shear stress has been previously demonstrated in endothelial cells. The remaining genes constitute known and uncharacterized species, many of which have not been described in vascular endothelium. The characterized genes fall into a limited number of functional categories that include stress response modifiers, genes involved in cellular metabolism, transcription factors and signaling molecules, regulators of the cell cycle, and growth factors. Immunohistochemistry and in situ hybridization on tissue microarrays were utilized to evaluate the in vivo expression of a number of these genes in the vascular endothelium. Preliminary analyses demonstrate that indeed these genes are expressed in the endothelium in vivo. Quantitative PCR analyses performed on a subset of genes has confirmed their shear stress regulation. The in vitro regulation of these genes by prolonged, steady laminar shear stress and their in vivo endothelial expression suggest that these transcripts represent a set of the genes responsible for the establishment and maintenance of endothelial phenotype in vivo. Current efforts are directed at by understanding guest on the April role(s) 4, of 2013 these genes in the vascular wall in vivo.
a-20 Arterioscler Thromb Vasc Biol. Vol 22, No 5 Lipid-Coated SPIO: Introducing a Novel Tracer for MR Imaging of Macrophages Infiltration in Vulnerable Atherosclerotic Plaque Maziar Azadpour, Chinnaswamy Jagannath, Daniel Chan, Ward Casscells III, James T Willerson, Morteza Naghavi. Center for Vulnerable Plaque Research at the University of Texas-Houston and the Texas Heart Institute, Houston, TX; Department of Molecular Pathology, University of Texas-Houston Health Science Center, Houston, TX; Division of Oncology, University of Colorado Health Science Center, Denver, CO P113 SPIO (super paramagnetic iron oxide) is a MRI contrast media in the form of nano-particles consisting a central core of iron oxide coated by colloidal polysaccharide mainly dextran. We have previously shown that dextran coated SPIO including the commercially available SPIO (Feridex) are avidly taken up by macrophages, however, their uptake was followed by a significant rise in intracellular oxidative enzyme activity mainly due to respiratory burst associated with digestion of dextran in macrophages. Here we report a novel liposomal SPIO. We hypothesize that lipid-coated SPIO particles enter into macrophages through a different pathway than dextran-coated SPIO allowing more uptakes with less oxidative stress. Method: Mouse peritoneal macrophages were isolated in the culture medium and incubated with different SPIOs for 4 hours and then washed. After 24 hours production of nitric oxide (NO) was measured in supernatant by Greiss reagent. In a second series of experiments fluorescentlabeled SPIO (FL-SPIO) was added to macrophages in the presence of two inhibitors of manose receptor, dextran and mannan. Intracellular retention of FL-SPIO was measured after 2 hours in four different groups, macrophages with SPIO, SPIO and dextran, SPIO and mannan and no SPIO as control. Results: See Figure 1 (p0.05). Also FL-SPIO studies showed that mannan a known inhibitor of mannose receptor significantly inhibited macrophage uptake of dextran coated SPIO but not lipid coated SPIO. Conclusion: Liposomal SPIO are promising candidate for MR contrast enhanced imaging of macrophage infiltration in vulnerable plaque. P114 Evidence of Macropinocytosis in Atherosclerotic Lesions of White Carneau Pigeons Nancy L Jones, Marie A Plyler, Dawn C Schwenke, Mark C Willingham. Wake Forest University School of Medicine, Winston-Salem, NC Macropinocytosis, occurs constitutively, but is up-regulated in macrophages (M) by growth factors and phorbol esters. Macropinosomes (MP), are classified as phase-bright, fluid-filled organelles forming primarily at the base of membrane ruffles (diameters 0.2 -5.0 m). Recently, AcLDL and OxLDL were shown to stimulate macropinocytosis and be internalized in part via MP by pigeon monocyte-derived M (Jones and Willingham, Anatomical Record, 255:57–68). In this study, we considered whether MP formed within M of atherosclerotic lesions. Lesions from the thoracic aorta immediately anterior to the celiac bifurcation of hypercholesterolemic random bred White Carneau pigeons were incubated in vitro with the fluid phase indicator Dextran-biotin. After a pre-incubation with buffer, aortic segments were incubated with Dextran-biotin (10,000 or 500,000 MW, 1 mg/ml) with either buffer, AcLDL (20 g protein/ml) or M-CSF (150 ng/ml) for 1 hr 37°C. Using peroxidase-streptavidin to detect the presence of biotin-dextran, we found that many M foam cells in the lesions had dextran-filled organelles consistent with MP 0.2 - 5.0 m. M foam cells within lesions incubated without AcLDL or M-CSF showed some MP, indicating a basal level of MP in lesion. Equivalent aortic segments incubated with either AcLDL or M-CSF had more Dextran-filled MP. This study demonstrates that macropinocytosis occurs in M within atherosclerotic lesions in vitro and suggests that macropinocytosis may occur in M within atherosclerotic lesions in vivo. Figure: In Vitro Macropinosome Formation in Lesions. Aorta was pre-incubated for 30 min, and subsequently, was incubated with dextran-biotin (500,000 MW) with AcLDL for 1 hr 37°C. Nnucleus, arrowMP. Bar10m Downloaded from http://atvb.ahajournals.org/ P115 Versican Content Is Dynamically Regulated during Expansion and Regression of Flow-Dependent Neointimal Thickening in Baboon Vascular Grafts Jens W Fischer, Richard D Kenagy, John Sandy, Stephanie Lara, Alexander W Clowes, Thomas N Wight. Institute of Pharmacology, University Kiel, Kiel, Germany; Division of Vascular Surgery, University of Washington, Seattle, WA; Shriners Hospital for Children-Research, Tampa, FL; Department of Pathology, University of Washington, Seattle, WA; Hope Heart Institute, Seattle, WA An experimental model of graft healing was used to analyze changes in the composition of extracellular matrix during expansion and regression of the neointima. In baboon aortoiliac grafts, the extent of neointimal thickening is dependent on the blood flow, which can be modulated experimentally by the construction of arteriovenous fistulas. In this model, normal flow induces neointimal thickening whereas high flow induces regression of this thickening. During neointimal expansion, the subendothelial part of the neointima is enriched in proteoglycans and stains intensely for versican. This versican-rich region also shows the highest proliferative activity of smooth muscle cells (SMC). In contrast, the deeper part of the neointima contains other components of the ECM as well such as type 1 collagen, biglycan and fibronectin and exhibited considerably less proliferative activity of SMCs. Neointimal regression in response to high flow was associated with a dramatic loss of proteoglycan as detected by histochemical staining. Furthermore, immunostaining with an antibody that recognizes a cleavage product of the core protein of versican shows intense staining of this region and little staining of the expanded neointima under normal flow. The present data support the hypothesis that accumulation or removal of proteoglycans, in particular versican, mediate adaptation of neointimal area and lumen size in response to changes in shear stress. Gene Knockouts for Dopamine Beta-Hydroxylase (DBH), 1B- and 1D-Adrenoceptors (AR) Suggest 1-AR-Mediated Trophic Action in Carotid Injury and Pulmonary Hypertension (PH) James E Faber, Caroline L Szymeczek-Seay, Susanna Cotecchia, Gozoh Tsujimoto, Hua Zhang. University of North Carolina, Chapel Hill, NC; University of Lausanne, Lausanne, Switzerland; Natl Children’s Med Res Ctr, Tokyo, Japan P116 In previous cell/organ culture & in vivo studies we have shown: norepinephrine (NE) has direct hypertrophic effects on smooth muscle cells & adventitial fibroblasts of rat aorta & carotid; this effect is strongly augmented after balloon injury to contribute significantly to wall growth, lumen loss, & inward remodeling; antagonist studies suggest mediation by 1A- & 1B- but not 1D-, 2D/A or -ARs that are also expressed in both media & adventitia (Zhang & Faber, Circ Res 89(8),2001; Erami et al, FASEB J 15 (5):A947,2001). Herein, a model of outward remodeling was used in knockout (KO) and wildtype mice. Carotid was over-distended by pressure, followed by air drying of endothelium. This causes increased (inc) lumen area, medial & adventitial area & thickness, and external elastic lamina circumference. DBH-KO prevented these inc, except for adventitial inc [50% less inc (p.05)]. 1B-KO prevented all inc. In contrast, 1D-KO only attenuated inc medial area & thickness (by 65%, p.05), possibly due to small decrease (dec) arterial pressure in 1D-KO only. To test for a NE trophic contribution to PH, mice were given 21days of 10% FIO 2. DBH-KO & 1B-KO did not significantly dec rt. ventricular systolic pressure (RVP) or hypertrophy (RVH), but hematocrit (HCT) inc WT in both KOs (p.05), suggesting less pulmonary vascular resistance (morphometry in progress). 1B involvement is consistent with induction of its promotor by hypoxia (Eckhart, Faber et al, PNAS 94:9487, 1997). 1D-KO dec RVP by 40% (p.05) & RVH by 50% (but p.05), while HCT inc similarly; morphometry should indicate if this reflects the small dec in arterial pressure in 1D-KO. These data suggest NE and 1-ARs may have a direct trophic role in injury-induced vascular remodeling. P117 A Role for Glutathione Reductase in OxLDL-Induced Macrophage Oncosis Reto Asmis, Jim G Begley. University of Kentucky, Lexington, KY Electron microscopy studies suggest that in human atherosclerotic lesions oncosis not apoptosis is the predominant mode of macrophage death. While macrophage apoptosis has been studied extensively, the mechanism of macrophage oncosis is poorly understood. We showed previously that oxidized LDL (OxLDL) induces oncosis in macrophages and macrophage-derived foam cells. Here we show that in human macrophages OxLDL promotes intracellular oxidative stress and caspase-3 activation within 4hofstimulation. Both oxidative stress and activation of caspase-3 continued to increase over 24 h. After 24 h, macrophage lysis was detected and continued to increase thereafter. Both OxLDL-induced oxidative stress and macrophage lysis, but not caspase-3 activation were inhibited by the water-soluble antioxidant Trolox. Conversely, neither the caspase-3 inhibitor z-DEVD-fmk or the general caspase inhibitor z-VAD-fmk blocked OxLDL-induced macrophage lysis, demonstrating that caspase activation was not required for OxLDL-induced macrophage oncosis. OxLDL also promoted the accumulation of oxidized glutathione and increased protein S-glutathiolation. Pretreatment of macrophages with low doses of the glutathione reductase inhibitor 1,3-bis[2- Chloroethyl]-1-nitrosourea (BCNU) in the absence of OxLDL did not induce cell lysis. However, BCNU pretreatment potentiated OxLDL-induced cell lysis and protein S-glutathiolation. Pretreatment of macrophages with high doses of BCNU increased levels of oxidized glutathione, promoted protein S-glutathiolation and induced cell lysis in the absence of OxLDL. Our results suggest that the oxidative inactivation of glutathione reductase may play an important role in OxLDL induced-macrophage by guest on April oncosis. 4, 2013
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