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

More documents

Recommendations

Info

severe reduction in HDL (90%). Hepatomegaly (liver/body ratio 3.6X) and lipid accumulation were evident. Lipoprotein analysis by FPLC confirmed that T0901317 treatment nearly eliminated HDL and caused a severe VLDL accumulation. Gene expression analysis of the LXR target genes SREBP1c and ABC1 showed that levels were unchanged in the liver and significantly induced in both the intestine and adipose tissue. Hepatic apo CIII, lecithin: cholesterol acyltransferase (LCAT) and hepatic lipase expression were reduced 69, 88 and 87% respectively, while both hepatic and intestinal apo A-I and apo CII expression were unchanged. Hepatic lipoprotein lipase (LPL) was increased (3.8X) while there were no changes in the expression of either LPL or hormone sensitive lipase in adipocytes. This data suggests that HDL production may be impaired due to decreased LCAT synthesis and that the hypertriglyceridemia may be due to either increased VLDL triacylglycerol production or decreased VLDL remnant uptake. Together, these results strongly suggest that a diabetic background may exacerbate the lipogenic effects of the LXR ligand T0901317 leading to a severe hypertriglyceridemia, hepatotoxicity and a resulting loss of plasma HDL. Expression of Scavenger Receptor BI in Macrophages Stimulates Esterification of Plasma Membrane Cholesterol Zhi H Huang, Theodore Mazzone. Rush Presbyterian and St Luke’s Medical Center, Chicago, IL SR-BI expression has been shown to facilitate the bi-directional movement of sterols between cells and extracellular acceptors; perhaps related to a reorganization of plasma membrane lipid domains. The aim of this study was to investigate whether SR-BI expression influenced the subcellular disposition of endogenous membrane cholesterol in macrophages. We constitutively expressed SR-BI in J774 macrophages or CHO cells at 4 and 7 fold higher levels compared to control cells. The rate of new cholesterol synthesis was higher in both macrophages and CHO cells (3.9, and 2.1 fold increase respectively, both p0.05), as a result of increased SR-BI expression; likely due to increased sterol efflux mediated by SR-BI. However, increased SR-BI expression also enhanced incorporation of 14 [C]oleate into cholesterol ester in J774 macrophages (2.6 fold increase, p0.05) but not in CHO cells. Experiments utilizing selective labeling of plasma membrane sterol with 3 [H]cholesterol at 15 o C indicated that the source of cholesterol for increased cholesterol ester synthesis in SRBI-expressing macrophages was derived from plasma membrane. Macrophages with increased SR-BI expression showed a 4.6 fold increase (p0.01) in 3 [H]cholesterol ester formation. There was no increase of plasma membrane cholesterol esterification in CHO cells with increased SR-BI expression. Addition of 25hydroxycholesterol to macrophages eliminated the difference in oleate incorporation into cholesterol ester between control and SRBI-expressing cells, but the increase of plasma membrane cholesterol esterification persisted. When acetate was used to monitor cholesterol ester synthesis, SR-BI expression increased synthesis (3.3 fold higher, p0.05) in macrophages and this increase was maintained after stimulation of sterol efflux with -CD (3.1 fold higher, p0.05). These results suggest that expression of SR-BI in macrophages facilitates the movement of cholesterol between plasma membrane and endoplasmic reticulum compartments. This enhanced transport could be related to the previously noted re-ordering of cholesterol within subdomains of cell plasma membrane. P97 Evidence for Matrix Metalloproteinases and Silent Plaque Rupture in the Development of Unstable Atherosclerotic Plaques in Male Apolipoprotein E Deficient Mice Jason L Johnson, Sarah J George, Andrew C Newby, Christopher L Jackson. University of Bristol, Bristol, UK The rupture of an atherosclerotic plaque with associated thrombosis is the main underlying cause of myocardial infarction and stroke. We aimed to determine the involvement of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of MMPs (TIMPs) during early plaque development and progression in our apolipoprotein E knockout mouse (ApoE -/- ) model of plaque rupture. Six-week-old male and female animals (C57/Bl6;Sv129) were fed a high-fat diet for 8 weeks and culled. Blood was taken for cholesterol analysis. Animals were perfusion fixed and the brachiocephalic artery, aortic arch, aortic root and heart were removed. Serial sections were examined by H&E, EVG, sirius red and immunocytochemistry for MMPs and TIMPs, smooth muscle cells (SMC), macrophages, MCP-1, T-cells and cleaved-PARP for apoptosis. In situ zymography was performed to detect MMP activity. Male mice had statistically greater total cholesterol levels when compared to females (451v202 mmol/l; p0.02). The brachiocephalic artery and aortic sinus of female mice (n6) contained small fatty streaks consisting of lipid-laden SMC-derived foam cells. Macrophage-rich atherosclerotic plaques were identified in the brachiocephalic artery, aortic sinus, aortic arch and coronary arteries of male mice (n12). 66.6% (8 out of 12) of brachiocephalic lesions exhibited signs of silent plaque rupture at shoulder regions, characterised by breaks in the internal elastic lamina and associated thrombus incorporation within the plaque. Increased MMP activity and MMP-3, -7, -9, -13 & -14, TIMP-2 & -3 expression was detected, predominantly in the macrophage rich shoulder regions. Foam cell emigration with associated thrombus, apoptotic cells and MCP-1 expression was also observed. Increased MMP activity, foam cell emigration, and apoptosis occur in silent rupture of early male ApoE -/- brachiocephalic atherosclerotic plaques. Healed and subsequent silent plaque ruptures may result in increased plaque burden and therefore, increase the propensity for occlusive plaque rupture as observed in this model. Downloaded from P96 http://atvb.ahajournals.org/ Poster Presentations a-17 P98 Oxidized Low Density Lipoprotein Upregulates CXCR4 Expression in Human CD4 T Cells Ki Hoon Han, Jong Min Song, Cheol Whan Lee, Jae Joong Kim, Seong Wook Park, Seung-Jung Park. Asan Medical Center, Seoul, South Korea Oxidation of low density lipoprotein and the infiltration of circulating CD4 T cells into the vascular wall are detected from the early stage of atherogenesis. The chemokine receptor CXCR4 is expressed in CD4 T cells within the peripheral blood and atheroma. Smooth muscle cells, endothelial cells and macrophages in human atherosclerotic plaques strongly express stromal derived factor (SDF-1) and the SDF-1 - triggered activation of CXCR4 induces the chemotaxis of T cells and possibly contributes to the T cell - mediated immunomodulation in the plaque. This study results demonstrated that the oxidized low density lipoprotein (OxLDL) upregulated CXCR4 expression in human CD4 T cells. The treatment of CD4 T cells isolated from the peripheral blood with OxLDL enhanced the amounts of both CXCR4 transcripts and proteins up to 4 fold in a dose (-10ug/ml) and time (-72hours) dependent manner. Among the components of OxLDL, lysophosphatidylcholine (lysoPC) showed the identical properties in the regulation of CXCR4 expression, whereas POVPC or KLH-conjugated phosphorylcholine did not affect the expression level of CXCR4. The positive regulatory effect of OxLDL and lysoPC on CXCR4 was nearly completely abolished by the incubation with CAPE (10 ug/ml), a NFkB inhibitor. The inhibition of tyrosine kinase and protein kinase C did not attenuate the effect of OxLDL on CXCR4 expression. Pretreatment of human CD4 T cells with lysoPC (10 ug/ml/24hrs) promoted the SDF-1 - induced migration 3 fold. The production of proinflammatory cytokines i.e. IL-2 and TNF-alpha from anti-CD3-immobilized CD4 T cells after SDF-1 costimulation was enhanced up to 4 fold by the pretreatment of the cells with lysoPC. Costimulatory effect of SDF-1 to increase surface expression of CD25 and CD154 (CD40 ligand) in anti-CD3-immobilized CD4 T cells was not obvious in this study. However, the activation of CXCR4 by SDF-1 significantly prevented the OxLDL - induced downregulation of CD28. These study results suggest that OxLDL in atherosclerotic lesion may directly and indirectly regulate the functional activity of CD4 T cells, which may play an important role in the progression of atherosclerosis. P99 Absence of CCR2 Receptors in Bone Marrow-Derived Cells Decreases Angiotensin II Induced Atherosclerosis and Abdominal Aortic Aneurysms in ApoE Deficient Mice Punnaivanam Ravisankar, Lisa A Cassis, Stephen Szilvassy, Alan Daugherty. University of Kentucky, Lexington, KY Angiotensin II (AngII) infusion promotes atherosclerosis and abdominal aortic aneurysm (AAA) formation in hyperlipidemic mice. AngII-induced vascular disease is characterized by increased arterial infiltration of monocytes and lymphocytes. A potential mediator of this infiltration is monocyte chemoattractant protein (MCP-1), a CC group chemokine that exerts its effect predominantly via CCR2 receptors. To determine the role of CCR2 in AngII-induced vascular pathology, we created chimeric apoE-/- x CCR2/ and apoE-/- x CCR2-/- mice by bone marrow transplantation. Eight week old male C57BL/6 ApoE-/- recipient mice were lethallyirradiated (900 rads) and transplanted with bone marrow from either CCR2/ or CCR2-/- C57BL/6 ApoE-/- donor mice (CCR2/ recipients n9; CCR2-/- recipients n12). Mice were permitted 6 weeks for bone marrow repopulation, then fed a high-fat diet and infused subcutaneously with AngII (1000 ng/kg/min) using Alzet osmotic pumps for 28 days. AngII infusion increased systolic blood pressure equally in CCR2/ and CCR2-/- groups. Serum concentration of total cholesterol was decreased by 21% (P0.009) in the CCR2-/- group, but there was no difference in triglycerides. Cholesterol was decreased in VLDL and LDL fractions in the CCR2-/- group. Serum MCP-1 concentrations were not different between the groups. CCR2 deficiency decreased AngII-induced atherosclerosis in both the aortic root (64%, P 0.002) and thoracic aorta (33%, P0.006). AAA formation was decreased in the CCR2-/- group (78% in CCR2/; 8% in CCR2-/-, P0.002). In conclusion, these data demonstrate that absence of CCR2 receptors in bone marrow derived cells reduces AngII-induced atherosclerosis and AAA in ApoE -/- mice. P100 Human (CETP X ApoB-100) Double Transgenic Mouse: Model of Profound Hyperinsulinemia, Obesity, Mild Hyperlipidemia and Limited Atherosclerosis Kathryn K McMahon. Texas Tech University Heatlh Sciences Center, Lubbock, TX Human hyperinsulinemic pre-diabetes are prone to atherosclerosis. Current hypotheses suggest this is due to increased vascular inflammatory responses to elevated blood lipid oxidation. Indeed, humans develop either or both type 2 diabetes and atherosclerosis with chronic relatively mild excesses of cholesterol and triglycerides. A mouse model, the human CETP X human ApoB-100 double transgenic mouse (dTrG), has been shown to develop type 2 diabetes, hyperinsulinemia, hyperlipidemias and hypertension. We hypothesized that this model would also develop atherosclerosis. To test this hypothesis, we fed male control (C57Bl/6) or dTrG mice a either a chow (4% fat/no cholesterol) or 34% fat/0.1% cholesterol (high fat) diet for 16 weeks (at least 3 mice/group). Blood samples were taken monthly to assay plasma insulin, glucose, total cholesterol and triglyceride levels. Body weights were determined weekly. At the end of the feeding regime, aortas and hearts were evaluated for atherosclerotic plaque formation. At the initiation of the feeding regimes, the dTrG mice had mildly elevated (i.e. less than 2-fold) insulin, glucose, and cholesterol levels and triglyceride levels were elevated by 3-fold. At the end of the feeding regime, dTrG fed chow had insulin levels over 6-fold higher than control mice fed chow and glucose, cholesterol and triglyceride levels were less than 2-fold higher than controls. Control mice and dTrG mice fed the high fat diet had insulin levels over 33- and 46-fold higher than the control/chow mice, respectively. The high fat-fed mice (control and dTrG) had glucose levels less than 2-fold above the control/chow mice, cholesterol by guest levelson of 3- April and 4-fold 4, 2013 above control/chow mice and triglyceride levels 2- and
a-18 Arterioscler Thromb Vasc Biol. Vol 22, No 5 4-fold above control chow mice. Body weights increased in both control and dTrG mice fed the high fat diet by 41% and 59% respectively. Atherosclerosis plaque formation was not different in the control or dTrG mice fed the chow or high fat diets. In conclusion, this dTrG mouse model is hyperinsulinemia, obese and mildly hypercholesterolemia but does not have elevated atherosclerotic plaque formation. P101 Transforming Growth Factor- and Fibronectin Expressions after Balloon Catheter Injury in Diabetic Rats and the Effects of Losartan Zheng L Li, Fu X Hua, Li C Guang, Wang Jun, Yi Lijing Q Xia. Second Affiliated Hospital of Hebei Medical University, Shijiazhuang, China; Institute of Pharmacology, Henan Medical University, Zhengzhou, China Aim: To study the mechanisms of the high restenosis rate of the patients with diabetes mellitus after percutaneous transluminal coronary angioplasty (PTCA) and the influences of losartan on excellular matrix constituents(EMC)formation and deposition. Materials and methods: The thoracic aortae of 16 diabetic rats induced by streptozotocin and 8 normal rats were injured by balloon. All rats were divided randomly into three groups:control ,diabetes mellitus(DM), and losartan. Losartan group were treated with losartan 20mg.kg -1 .d -1 . 2 weeks after the balloon injury, all thoracic aortae were excised. Transforming growth factor receptors (T R)1 ,2 and fibronectin(FN)were determined by RT-PCR ,Northern blotting and immunohistochemical analysis. HPLC-RIA was used to measure angiotensin(Ang)2 level in artery. Results: We found that expressions of T R2,FN mRNA and protein in DM group is increased by 20%,21%,32%,45% respectively than control group.The level of Ang 2 in DM group was enhanced by 22% than control group. There was no difference in the expression of T R1mRNA and protein between control and DM groups. Administration of losartn could reduce expressions of T R2, FN mRNA and protein by 60%,53%,47%,65% respectively than DM group.It also could reduce the level of Ang 2 by 36% than DM group. Conclusions: It is suggested that there is an interaction between T R2and the pathogenesis of diabetic restenosis after PTCA. Losartan can suppress the expression of T R2 in artery. P102 Evaluation of Carotid Calcification as a Screening Marker for Coronary Artery Stenoses Jörg Nossen, Thomas Vierzigmann, Erich Lang. Waldkrankenhaus St. Marien, Erlangen, Germany Background: Ultrasound is an established and widespread method for examination of carotid arteries concerning calcified plaques and stenoses. Questionable is if hard plaques in the cerebral arteries are a surrogate screening marker for stenoses of coronary arteries. Methods: 139 patients underwent cardiac catheterization with selective coronary angiography (technique of Judkins) and ultrasound examination (B-mode) of carotid arteries. In case of calcified plaques number and distribution among the extracranial cerebral arteries were measured. Coronary angiograms were analyzed for disease severity and extent (number of main vessels with 50% stenosis). Conclusions: Ultrasound of carotid arteries is a simple screening marker to improve predictive value of risk factor-based multivariate models. Circulation Is Established in a Step-Wise Pattern in the Mammalian Embryo Kathleen E McGrath, Anne Koniski, James Palis. University of Rochester, Rochester, NY P103 The common site and time of origin of the first embryonic endothelial and blood cells within the yolk sac suggests that hematopoietic and vascular fates are closely intertwined. Furthermore, the forming vascular system is the site of blood synthesis in the early embryo. To better understand the relationship between the hematopoietic and vascular systems, we investigated the establishment of the circulation in mouse embryos by examining various vascular beds for the presence of yolk sac-derived primitive erythroblasts and definitive hematopoietic progenitors. Our studies reveal that small numbers of erythroblasts first enter the embryo proper at 6–8 somite pairs (sp) (embryonic day 8.25 (E8.25)) which correlates with the proposed onset of cardiac function. Hours later (E8.5), increasing numbers of red cells have entered the embryo proper, however, there is still a predominance (10-fold greater number) of erythroblasts in the yolk sac. The number of red cells continues to expand in the embryo proper reaching a steady state of approximately 40% of total cells between 26–30 sp (E9.75). During this early stage of circulation (between 6 and 25 sp), erythroblasts are not distributed throughout the embryo’s vasculature, rather they gradually progress though specific vascular beds. The establishment of a robust circulation at E9.75 correlates with vascular remodeling suggesting that vessel arborization and/or smooth muscle recruitment are required. We also examined the distribution of committed hematopoietic progenitors within the developing mouse conceptus. Prior to E8.25, all progenitors were found in the yolk sac. When normalized to circulating erythroblasts, there was still a significant enrichment (20 to 5-fold) of progenitors in the yolk sac compared to the embryo proper from E9.5 to E10.5. These results suggest that the yolk vascular network remains a site of progenitor Downloaded synthesis and/or from preferential adhesion http://atvb.ahajournals.org/ even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular system develops gradually and that specialized vascular-hematopoietic environments exist after circulation becomes fully established. Circulating Activated Platelets Promote Leukocyte Recruitment to Atherosclerotic Lesions of Atherogenic Mice Yuqing Huo, Klaus Ley. University of Virginia, Charlottesville, VA P104 Occurrence of activated circulating platelets and platelet-leukocyte aggregates in the individuals with atherosclerosis has been found for several decades. However, it is unknown whether they are just makers or active players. Here, we investigated the roles of activated platelets in the interactions of leukocytes with atherosclerotic lesions of apoE-/- mice in a novel in vivo model. ApoE-/- mice fed with western diet for 8 weeks were able to develop atherosclerotic lesions on the external branch of carotid arteries. Using epifluoresence intravital microscopy, it was rare to observe leukocytes labeled with rhodomine 6G interacting with atherosclerotic lesions, consistent with chronic inflammation-the nature of atherosclerosis. Interactions of leukocytes with atherosclerotic lesions were dramatically promoted following perfusion of activated mouse or human platelets but not the resting ones. These interactions occur mainly on early atherosclerotic lesions, shoulders, but not on the centrals of established lesions. Blockade of endothelial P-selectin inhibits leukocyte adhesion on atherosclerotic lesion by 55% while blocking platelets P-selectin almost completely prohibited these interactions mediated by activated platelets. Activated platelets also were not able to cause leukocytes deficient in P-selectin ligand, PSGL-1 to interact with atherosclerotic lesions. Examination using SEM showed that the number of leukocytes on atherosclerotic lesion after activated platelet perfusion was 30–50 times higher than that after perfusion of activated platelets without P-selectin. Platelets were found to bind on most of the leukocyte adhering on atherosclerotic lesions. These results suggest that activated platelets can serve as active players in atherosclerosis. Molecules including platelet P-selectin and leukocyte PSGL-1 are crucial for the involvement of activated platelets in atherosclerosis. The findings clarify the molecular mechanism involved in contribution of platelets to atherosclerotic lesions and suggest potential new approaches to curbing the development of atherosclerosis lesions. P105 Endotoxin Activates Pro-Inflammatory Cytokine and Superoxide Production in Human Blood Vessels: Relevance to Atherosclerosis James B Rice III, Lynn Stoll, Wei G Li, Neal L Weintraub. University of Iowa Hospitals and Clinics, Iowa City, IA Atherosclerosis, the leading cause of death worldwide, is a chronic inflammatory disorder. The sources of vascular inflammation in atherosclerosis, however, remain to be elucidated. One potentially important source of vascular inflammation is endotoxin, a cell wall component of Gram-negative bacteria. Plasma endotoxin level of 50pg/ml has recently been identified as a powerful, independent risk factor for the development of atherosclerosis. Potential mechanisms whereby endotoxin could contribute to atherosclerosis include increased production of superoxide and pro-inflammatory cytokines. Accordingly, human saphenous vein explants were treated with low levels of endotoxin (10 ng/ml). Release of the cytokines interleukin-8 (IL-8) and monocyte chemoattractant peptide-1 (MCP-1), key in recruitment of inflammatory cells to atherosclerotic lesions, was measured by ELISA. To detect superoxide in situ, tissues were stained with hydroethidine (HE) and examined by laser confocal microscopy. Measurable increases in IL-8 and MCP-1 release were observed at endotoxin concentrations as low as 30 pg/ml, well within the range of levels that have been associated with an increased risk for atherosclerosis. Treatment with 100 pg/ml endotoxin resulted in approximately 7-fold increase in IL-8 release over basal levels; similar results were obtained in cultured human coronary artery smooth muscle cells. HE fluorescence was dose-dependently increased throughout the vessel wall by a 4-h incubation with 1–10 ng/ml endotoxin, indicating increased superoxide levels. We conclude that clinically-relevant concentrations of endotoxin activate inflammatory cytokine and superoxide production in human blood vessels, suggesting a putative mechanistic link between endotoxin, vascular inflammation, and atherosclerosis. P106 In Baboon Smooth Muscle Cells, Interleukin-1 Inhibits PDGF-BB Induced Migration through a Cox2 Dependent Pathway Michael J Englesbe, Jessie Deou, Alexander W Clowes, Guenter Daum. University of Washington, Seattle, WA Objective: Interleukin-1 (IL-1) is a proinflammatory cytokine that is present in the injured and atherosclerotic vessel wall. Since PDGF plays an important role in the pathogenesis of vascular injury, we studied the effects of IL-1 on PDGF-BB induced baboon smooth muscle cell (SMC) migration. Methods: SMC migration was assayed with a modified Boyden microchemotaxis chamber, as previously described. Data is expressed as the fold increase in migration relative to controlSEM. Findings: IL-1 (0.1ng/ml) inhibits PDGF-BB (10ng/ml) induced migration (IL-1 PDGF BB: 2.230.10 vs. PDGF BB control: 3.800.12). This IL-1 induced inhibition is markedly attenuated by treatment with a Cox2 specific inhibitor, NS398 (10M) (PDGF BB NS398: 3.260.27 vs. PDGF BB IL-1 NS 398: 3.040.10). The IL-1 effect is also relieved by the p38-MAP kinase inhibitor, SB20358 (3M) (PDGF BB SB20358: 3.190.23 vs. PDGF BB IL-1 SB20358: 3.550.40), indicating that Cox2 induction may be mediated by p38-MAP kinase. By Western blot analysis we demonstrate that IL-1 mediated Cox2 induction is inhibited by SB20358. Conclusion: IL-1 inhibits PDGF-BB induced SMC migration. This inhibition occurs through IL-1 induced Cox2 expression via a p38-MAP by kinase guest dependant on April signaling 4, 2013 pathway.
Page 1 and 2: 3rd Annual Conference on Arterioscl
Page 3 and 4: a-2 Arterioscler Thromb Vasc Biol.
Page 17: a-16 Arterioscler Thromb Vasc Biol.
Page 69 and 70:
a-68 Arterioscler Thromb Vasc Biol.
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77 and 78:
Page 79 and 80:
show all

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

Create successful ePaper yourself

Delete template?

Save as template?