Oral Presentations - Arteriosclerosis, Thrombosis, and Vascular ...

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the increased release of superoxide from the cells. The changes caused by elevated glucose were associated with loss of zinc content in eNOS and were prevented by a NOS inhibitor or superoxide dismutase, indicating that they were caused by peroxynitrite. Furthermore, eNOS purified from diabetic low density lipoprotein (LDL) receptor deficient mice, which showed an increased aortic lesions, had decreased NO synthetic activity, lowered zinc content, and decreased enzyme-active eNOS dimers resulting in an increased thiol oxidation and increased generation of oxidants. These observations indicate that in diabetes eNOS is oxidized by peroxynitrite resulting in an enzymatic uncoupling and generation of oxidants contributing further to the oxidant stresses in these cells. The principal mechanism appears to be oxidation of the zinc-thiolate complex that maintains the enzyme as active dimers rather than via oxidation of BH 4. Oxidation of zinc-thiolate cluster of eNOS, therefore, provides a novel mechanism for modulation of the enzyme function in vivo. Selectin-Like Bond Kinetics Promote Rapid Platelet Adhesion: The GPIb-vWF Bond and the Impact of Type 2B Mutations Thomas G Diacovo, Gaurav Girdhar, Avril Lawshe, David W Schmidtke, Ian J Laurenzi, Scott L Diamond, Teresa A Doggett. Division of Newborn Medicine, Washington University, St. Louis, MO; Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA Rapid bond formation and dissociation are characteristic of the selectin family of adhesion receptors that promote the capture and translocation of leukocytes on vascular endothelium. In contrast selectins, the platelet glycoprotein receptor Ib alpha (GP Ib) mediates plateletvessel wall interactions by binding to the A1 domain of surface-immobilized von Willebrand factor (vWF-A1). This receptor-ligand pair, however, has been reported to have slow intrinsic bond kinetics. We now present evidence that the GPIb-vWF-A1 bond has properties previously identified with selectins: flow dependent adhesion and rapid and force-dependent bond kinetics. Characterization of GPIb interactions with plasma vWF or recombinant wt A1 protein in vitro demonstrates that a critical level of hydrodynamic flow is required to initiate adhesion between this receptor-ligand pair. The dissociation rate constants, koff, for the GPIb vWF-A1 bond, as determined by analysis of pause times, were found to vary exponentially (4.2 0.8 s-1 to 6.9 0.3 s-1) as a function of the force applied to the bond (from 36 to 217pN). The physiologic significance of bond mechanics in platelet adhesion was exemplified by type 2B von Willebrand disease (vWD), a bleeding disorder resulting from the spontaneous binding of plasma vWF to circulating platelets. Kinetic analysis of mutations associated with this disorder revealed a loss of the shear threshold phenomenon, a 6-fold reduction in the intrinsic koff, and a 2-fold increase in the reactive compliance of the bond suggesting a greater propensity to breakage in response to hydrodynamic force. Thus, the increased bond lifetime observed for type 2B vWF and the concomitant loss of the hydrodynamic threshold requirement for adhesion demonstrate the deleterious consequences that can result from an alteration in the biomechanical regulation of a receptor-ligand pair. Importantly, our results indicate that evaluation of receptor-ligand interactions under physiological relevant conditions is paramount to understanding how biomechanical properties of bonds ultimately control the process of cell adhesion. Increased Atherosclerosis in Apo E Deficient Mice Lacking Macrophage-Derived ACAT1 Yan Ru Su, Dwayne Dove, Amy Major, Alyssa Hasty, Robert Falese, Macrae Linton, Sergio Fazio. Vanderbilt University Medical Center, Nashville, TN Acyl-coenzyme A: cholesterol acytransferase (ACAT) is an enzyme responsible for esterification of free cholesterol to its intracellular storage form, cholesterol ester, and therefore plays an important role in cellular cholesterol homeostasis. Two isoforms of ACAT have been cloned in mammals: ACAT1 is preferentially expressed in macrophages, adrenal gland and kidney while ACAT2 is mainly expressed in liver and intestine. ACAT1 catalyzes the esterification of free cholesterol in macrophage leading to intracellular cholesterol ester accumulation, foam cell formation therefore play a crucial role in early development of atherosclerotic lesions. Many studies have shown that nonselective inhibition of ACAT reduces atherosclerosis. However, previous study in our laboratory have shown that complete inhibition of ACAT1 leading to an increase in atherosclerosis in LDL receptor deficient mice, raising the concern that the complete absence of ACAT1 in macrophage can promote atherosclerotic lesion formation. The objective of our current study was to determine the effect of absence of macrophage-derived ACAT1 on atherosclerosis in apo E deficient mice using bone marrow transplantation. Our results show that the lack of ACAT1 in apo E deficient macrophages also promotes atherosclerotic lesion formation (89606/-46799 m 2 in ACAT1 and apoE double knockout mice, n9; compared to 50749/-28846 m 2 in apoE deficient mice n8, p0.05). Surprisingly, studies in peritoneal macrophages isolated from ACAT1 deficient mice showed a 30–40% reduction (p0.001) in cholesterol efflux compared to control macrophages. In conclusion, deficiency of macrophage ACAT1 appears to have deleterious consequences to vascular health in apoE-null animal model, suggesting that caution should be exerted in the developing of ACAT inhibitors aimed at treating human atherosclerosis. 24 25 26 that hepatic cholesterol synthesis and hepatobiliary transport are not affected in mice lacking ABCA1, findings incompatible with current concepts of RCT. ABCA1 is highly expressed in peripheral tissues, including macrophages, but also in liver and intestine: its function in these organs is unclear. Expression of the Abca1 gene is controlled by the liver X-receptor (LXR). We have evaluated the physiological effects of LXR activation on hepatic cholesterol metabolism, with special reference to hepatobiliary transport. For this purpose, Abca1 -/- and wildtype mice were treated with the LXR agonist T0901317 (10 mg/kg, 5 d). Plasma cholesterol levels increased in Abca1 -/- mice (1.11 vs. 0.50 mM) and in control mice (1.64 vs. 1.12 mM) upon treatment. In Abca1 -/- mice, the increase was exclusively in VLDL-sized fractions, whereas in wildtype mice VLDL and HDL cholesterol increased. Hepatic triglycerides were massively elevated in both strains after treatment, presumably related to induction of lipogenic genes. LXR activation strongly affected bile composition. Bile flow was identical in both strains, but bile salt excretion was diminished in treated wildtype (-22%) and in Abca1 -/- (-46%) mice. Likewise, biliary phospholipid output decreased upon treatment in both strains, in spite of unaffected expression of Abcb4, encoding the hepatic phospholipid translocase. In contrast, cholesterol output was significantly induced by treatment, leading to increases in cholesterol:phospholipid ratios from 0.13 to 0.46 and from 0.12 to 0.42 in wildtype and Abca1 -/- mice, respectively. Expression of Abcg5 and Abcg8, recently implicated in cholesterol transport, was induced in livers of treated mice. In conclusion, chronic activation of LXR in mice leads to enhanced hepatobiliary cholesterol removal independent from (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in the liver 27 Naturally Occurring Mutations of the Tangier Disease-Associated Protein, ABCA1, Prevent the Movement of Cholesterol to the Exofacial Side of the Plasma Membrane Ashley M Vaughan, John Oram. University of Washington, Seattle, WA The ATP-binding cassette protein, ABCA1, is involved in the apolipoprotein AI-mediated efflux of excess cholesterol from cells and is mutated in patients with Tangier disease. The lack of a functional ABCA1 protein in Tangier patients causes the absence of high density lipoprotein, and subsequently, an increased risk for cardiovascular disease. We have recently shown that over-expression of ABCA1 in cells leads to an increase in cholesterol oxidase-sensitive cholesterol on the exofacial side of the plasma membrane. To examine the effect of naturally occurring ABCA1 point mutations on protein function, we used site-directed mutagenesis to construct ABCA1 mutants which we then expressed in cells. Four mutants were constructed, two in the large amino-terminal extracellular loop of the protein, and one from each of the two ATP-binding domains. All four mutants were expressed at high levels in our cell system. In every case, expression of the mutant protein, when compared to wild-type ABCA1, lead to a significant decrease in both apolipoprotein AI binding and apolipoprotein AI-mediated cholesterol efflux from cells. Expression of all four mutants also failed to cause an increase in cholesterol oxidase-sensitive cholesterol on the exofacial side of the plasma membrane. It is tempting to hypothesize that the movement of cholesterol to the exofacial side of the plasma membrane in ABCA1 expressing cells is essential for the subsequent apolipoprotein AI binding and apolipoprotein AI-mediated cholesterol efflux. Thrombin Enhances Inflammation during Cardiac I/R Injury Oral Presentations a-5 Rafal Pawlinski, Craig Hampton, Jeanette Ennis, Patricia Andrade-Gordon, Edward Verrier, Timothy Pohlman, Nigel Mackman. The Scripps Research Institute, La Jolla, CA; University of Washington, Seattle, WA; R.W. Johnson Pharmaceutical Research, Spring House, PA Ischemia-reperfusion injury has many features in common with inflammatory responses and leads to endothelial dysfunction, microvascular collapse and impairment of blood flow, myocardial infarction and cell death. We and others have shown that the coagulation protease cascade contributes to injury during cardiac I/R injury. We have employed both rabbit and mouse models of cardiac I/R injury. Inhibition of either tissue factor (TF) or thrombin reduced infarct size whereas defibrinogenerating rabbits did not reduce infarct size. We measured the expression of various inflammatory mediators in the rabbit and mouse models. I/R injury induced the expression of transcription factors, such as Egr-1, adhesion molecules, such as ICAM-1, and chemokines, such as IL-8, MCP-1 and MIP-2. We also examined chemokine mRNA expression in mice subjected to different times of ischemia (5–30 min) or a fixed time of ischemia (30 min) with different times of reperfusion (30 min, 1, 2, 3 and 4 hours) to determine the kinetics of induction. Importantly, inhibition of thrombin in I/R-injured rabbits reduced MCP-1 expression and PMN infiltration. Finally, we used PAR-1 knockout mice to test the hypothesis that thrombin enhancement of inflammation was mediated by PAR-1. We observed a 43% decrease (p0.01) in infarct size in PAR-1-/- mice (mean infarct /- SE, 20 /- 4, n6) compared with PAR-1/ mice (mean infarct /- SE, 35 /- 3, n6). Taken together, these results indicate that the TF-thrombin-PAR-1 signaling pathway contributes to inflammation in cardiac I/R injury. Prevention of Diabetes-Induced Microangiopathy by Human Tissue Kallikrein Gene Transfer Increased Hepatobiliary Cholesterol Transport by Activation of the Liver Costanza Emanueli, Bonaria Salis, Paolo Madeddu. National Laboratory INBB, Osilo, Italy X-Receptor LXR Is Independent of ABCA1 in Mice Microvascular remodelling and insufficiency contributes to end-organ damage in diabetes. The Torsten Plösch, Tineke Kok, Vincent W Bloks, Rick Havinga, Martin J Smit, Giovanna lack of effective therapy for the treatment of microangiopathy urges to seek new strategies of Chimini, Albert K Groen, Folkert Kuipers. Department of Pediatrics, University Hospital vascular protection and repair. We evaluated if human kallikrein (HK) gene therapy can prevent Groningen, Groningen, Netherlands; Centre d’Immunologie, INSERM-CNRS, Marseille, or rescue microangiopathy in limb skeletal muscle in streptozotocin (STZ)-induced diabetic France; Department of Gastroenterology, Academic Medical Center, Amsterdam, Netherlands mice. We found that, after an initial increase in vascularity, diabetic mice develop progressive capillary and arteriole loss and rare faction, due to apoptotic cell death. This was associated The ATP binding cassette transporter ABCA1 is essential for HDL formation and therefore with remodelling of arterioles of various luminal diameter, impaired response to acetylcholine considered rate-controlling for reverse cholesterol Downloaded transport (RCT). Recently, from http://atvb.ahajournals.org/ we demonstrated or reperfusion, by guest shortage on ofApril muscular 4, cGMP 2013content and ultimately muscular damage. Chronic 28 29
a-6 Arterioscler Thromb Vasc Biol. Vol 22, No 5 insulin supplementation ensured adequate control of hyperglycemia and preserved in part endothelium-dependent vasodilation, yet did not prevent vascular pathology. In contrast, application of gene therapy with HK at early phases of diabetes (14 days) halted the progression of microangiopathy, as a result of neovascularization and inhibition of apoptosis. The beneficial action occurred no matter mice had been treated or not with insulin, which indicates that HK is effective independently from imposed metabolic control of the disease. Vascular effects of HK were limited to the site of injection, thus discounting the risk of inappropriate vascular growth in distant organs. The improved limb microcirculation allowed for an accelerated hemodynamic recovery during post-ischemic reperfusion. Finally, we documented that application of HK gene therapy at a later stage (60 days) with the attempt to rescue established microangiopathy results in a marked increase in muscular capillarity, which implies vascular regeneration. Our discoveries indicate that HK may be a useful tool for the treatment of microvascular disease in diabetics. 30 Megakaryocyte Endocytosis and Intracellular Trafficking of Plasma-Derived Factor V Results in a Physically Unique Platelet-Derived Cofactor Weston R Gould, Paula B Tracy. University of Vermont College of Medicine, Burlington, VT Platelet-derived factor Va (FVa) originates from plasma via megakaryocyte endocytosis of circulating factor V (FV). Despite the identical site of synthesis, platelet-derived FVa is a unique substrate for several enzymes. For example, in contrast to the plasma-derived cofactor, platelet-derived FVa is resistant to phosphorylation at Ser692 catalyzed by casein kinase II (CKII). These data suggest that, subsequent to its endocytosis by megakaryocytes, specific intracellular trafficking events lead to modifications that yield a cofactor resistant to phosphorylation at this site. Consequently, purified plasma and platelet-derived factor Va heavy chains (HC) were isolated by SDS-PAGE and subjected to digestion with trypsin such that the resulting fragments could be identified by MALDI-TOF mass spectroscopy. Such analyses allowed the identification of a fragment with m/z of 2087.8 corresponding to residues 685–701 (LEPEDEESDADYDYQNR) in the plasma-derived FVa HC, which, subsequent to phosphorylation by CKII, lead to a 80 Da shift of this fragment directly demonstrating the incorporation of phosphate into Ser692. Despite its resistance to phosphorylation at this site, MALDI-TOF analysis of the platelet-derived cofactor demonstrated that Ser692 was not modified suggesting that, post-endocytosis, any alteration to the protein that inhibits phosphorylation, must occur at an alternative location. Additional analyses of the heavy chain of platelet-derived FVa identified a fragment at m/z of 679.5 unique to the platelet-derived molecule that corresponds to the addition of an N-acetylglucosamine (GlcNac) at Thr402 (DTLK GlcNac) indicating for the first time, that the platelet-derived cofactor is physically distinct from the plasma-derived molecule although the functional consequence of this alteration has yet to be defined. However, the current study offers strong support of the concept that subsequent to endocytosis by megakaryocytes, plasma-derived FV undergoes retrograde transport to the trans-Golgi network and is retailored post-translationally to yield a unique platelet-derived cofactor. 31 HoxB5 Transcriptionally Regulates the Vascular Endothelial Growth Factor Receptor KDR/flk-1 In Vitro and In Vivo Yaxu Wu, Cam Patterson. Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, Chapel Hill, NC KDR/flk-1 is a receptor tyrosine kinase that participates in angiogenesis by serving as a receptor for vascular endothelial growth factor. It is also the earliest known marker for hemangioblasts and is required for endothelial and hematopoietic differentiation during development. We have studied the regulation of KDR/flk-1 as a model for understanding transcriptional events that control angiogenesis and endothelial cell differentiation. Using DNase I footprinting and gel shift analyses, we identified a novel AT-rich 20-bp sequence in the first intron of the mouse KDR/flk-1 gene that was bound by nuclear proteins in an endothelial cell-specific fashion. Using transgenic mice, we demonstrated that a 4-bp mutation in this element abolished endothelial-specific expression of a reporter gene under control of the KDR/flk-1 promoter/enhancer. We used a yeast one-hybrid approach to clone the nuclear protein(s) responsible for binding to this element. 5 positive clones were identified in a screen of a mouse embryo library with this element; 2 of these clones encoded the homeodomain protein HoxB5. HoxB5 bound to the wild-type KDR/flk-1 element, but not to a mutated sequence, demonstrating the specificity of this interaction. In transient transfection assays, HoxB5 was able to transactivate the KDR/flk-1 promoter 8–10 fold. Until now, the function of HoxB5 has been largely obscure. Our present results provide strong evidence that HoxB5 is required for KDR/flk-1 expression in vivo and in vitro, via a novel cis-acting element. This unexpected finding suggests that this homeodomain protein is a likely candidate transcription factor that regulates hemangioblast differentiation and angiogenesis. Bmx Mediates TNF-Induced Inflammatory Angiogenesis through Association with TNFR2 Ping An, Wang Min. University of Rochester, Rochester, NY As a proangiogenic factor, TNF promotes inflammatory angiogenesis in progression of rheumatoid arthritis through up-regulation of a large number of angiogenic factors such as VEGF, bFGF and MCP-1. However, the signaling of TNF-induced angiogenesis is not fully understood. Bmx/Etk, a member of Btk/Tec non-receptor tyrosine kinase family is highly expressed in endothelial cells and exerts a variety of bioactivities related with proliferation and migration through interaction with various signal complexes. Recently we showed that activity of Bmx, JNK kinases and JNK-dependent gene expression were increased dramatically in arthritic joints of hTNF transgenic mice. These data prompted us to examine the role of Bmx in inflammatory angiogenesis. Here, we showed Downloaded that Bmx constitutively from interacts with and is 32 http://atvb.ahajournals.org/ activated by TNFR2 but not TNFR1. In contrast, the dominant negative form of Bmx (Bmx-DN) abolishes activation of JNK induced by TNF, but not by overexpression of TRAF2, indicating that Bmx and TRAF2 activate JNK through different pathways. Overexpression of both the wild type (WT) and the constitutively active (SK) Bmx activate JNK, while the dominant-negative form (DN) and the kinase domain deletion mutant (DK) of Bmx inhibits TNF-induced JNK activation. Mapping studies show that SH3 domain in Bmx and the 16 amino acids within the C-terminus of TNFR2 (non-TRAF2-binding domain) are required for the interaction of the two proteins. We further investigated the biological activities of Bmx with two in vitro angiogenic models: a three-dimensional culture in collagen gel and a migration assay of EC. Results show that Bmx-SK facilitated, while Bmx-DK blocked, TNF-induced tube formation and “wound healing” of EC. Taken together, our data suggest Bmx is a critical mediator involved in TNF-induced angiogenesis and may provide a novel approach for treatment of angiogenesis dependentdiseases such as atherosclerosis, cancer and rheumatoid arthritis. Decreased Mural Cell Proliferation Is Associated with Impaired Vascular Development in the NG2 Knockout Mouse Ugur Ozerdem, William B Stallcup. The Burnham Institute, La Jolla, CA Confocal imaging with endothelial (CD31, flk-1, CD105), pericyte (alpha-SMA, PDGF betareceptor), cardiomyocyte (MLC2a, MLC2v), and nuclear markers was utilized to compare wild-type (wt) and NG2-knockout (ko) mice to reveal a functional role for the NG2 proteoglycan in cardiovascular development and hyperoxia-induced retinal angiogenesis. BrdU labeling was used to quantify proliferation of vascular cell types. We find that retinal angiogenesis is reduced in the ko mouse. At P17 the mean number of vascular nuclei in ischemic angiogenic retinal tufts is 54.9 per histological section in the ko retina versus 119.8 per section in the wt retina (p0.0019 for 10 wt eyes (50 sections) and 10 ko eyes (50 sections)). Decreased vascular cell proliferation was noted: 38.3% of endothelial cells incorporate BrdU in wt mice versus 22.8% in ko mice (p0.0147); 45.2% of wt pericytes label with BrdU versus 18.7% in ko mice (p0.0068). The endothelial cell/pericyte ratio is 1.161 in wt eyes versus 4.065 in ko eyes (p0.0011 for 2 wt eyes (10 sections) and 2 ko eyes (10 sections)). At E10 the NG2-null heart is distinguished from wt heart by a 46.4% ventricular enlargement (p0.0068), a 114.6% increase in the ratio of inflow tract (IFT)/outflow tract (OFT) diameter (p0.0001), and a 64% increase in the ratio of IFT/OFT wall thickness (p0.0039). Trabeculation is reduced in the ko ventricle. In wt ventricle, 67.6% of cardiomyocytes label with BrdU versus 26.9% in ko ventricle. In wt OFT, 53.5% of cardiomyocytes label with BrdU versus 27.6% in ko OFT. In wt IFT, 27.5% of cardiomyocytes are BrdU-positive versus 33.6% in ko IFT. This pattern is significant, since ventricle and OFT normally exhibit high levels of NG2 expression, while IFT is low in NG2. Conclusions: NG2-null mice exhibit diminished angiogenesis, decreased BrdU uptake by vascular cells (especially pericytes), and reduced pericyte investment of endothelial tubes. A larger but less trabeculated ventricle and a decrease in OFT size in the ko embryo may be due to reduced cardiomyocyte proliferation in these areas. Massive Xanthomatosis and Widespread Tissue Accumulation of Macrophages in Hyperlipidemic Mice Lacking ABCA1 Robert J Aiello, Omar L Francone, Dominique J Brees, Patricia-Ann Bourassa, Lori J Royer, Saralyn Lindsey, Timothy M Coskran. Pfizer Inc, Groton, CT The discovery of ATP-Binding Cassette Transporter A1 (ABCA1) and its role on High density lipoproteins (HDL) biogenesis has brought considerable attention to the potential for enhancing its activity as a therapeutic means to affect atherosclerosis. Despite the complete absence of HDL and tissue accumulation of macrophages in homozygous patients with Tangier Disease and mice lacking Abca1, the contribution to Abca1 to the development of atherosclerosis remains unclear. In this study we have examined whether the absence of Abca1 is accompanied by an increased susceptibility to atherosclerosis. In the setting of severe hypercholesterolemia caused by a deficiency of apolipoprotein E (apoE-/-) mice, the additional absence of Abca1 led to a marked alteration on the distribution of tissue macrophages with prominent accumulation in skin, resulting in severe xanthomatosis. Complete necropsy demonstrated age-related infiltration of macrophages in the uterus, stomach, lymph nodes, kidneys and lungs. The absence of ABCA1 did not affect the development and progression of atherosclerotic lesions in mice fed a chow or high fat diet despite the near complete absence of HDL and apolipoprotein AI (apoAI). Oxidized LDL and 12/15-Lipoxygenase Stimulate Actin Polymerization in Macrophages and Affect Phagocytosis of Apoptotic Cells Yury I Miller, Joseph L Witztum. University of California, San Diego, La Jolla, CA Atherosclerotic tissue is characterized by accumulation of oxidized LDL (OxLDL) and by many apoptotic cells despite the presence of macrophages, which normally efficiently engulf these moieties via scavenger receptors. Actin polymerization (formation of F-actin) is necessary for phagocytosis. Macrophages in murine atherosclerotic lesions express high levels of 12/15lipoxygenase (LO). Thus, we examined if LO and OxLDL could affect macrophage actin polymerization and phagocytosis. Digital deconvolution microscopy was used to image LO and F-actin in macrophages, and FACS was used to quantify F-actin levels. When macrophages were exposed to apoptotic cells, LO translocated from the cytosol to the cell surface and colocalized with F-actin of emerging filopodia. Inhibition of the LO activity by PD 146176 or disruption of the 12/15-LO gene down regulated F-actin formation in response to apoptotic cells, as shown by FACS analysis. Addition of LO products, such as 13(S)-HODE provoked F-actin formation in vitro and in cell lysates. When macrophages were incubated with OxLDL or minimally modified LDL (mmLDL), instead of apoptotic cells, enhanced actin polymerization was also observed. The levels of F-actin were increased by 20% in 10 minutes and 100% in 8 hours. by Morphologically, guest on April the macrophages 4, 2013 appeared extensively spread. In contrast to 33 34 35
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