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and also in 6 patients on statins with no muscle complaints. Abnormal muscle biopsies were attributed to statin toxicity if they demonstrated pathology consistent with mitochondrial dysfunction such as ragged red fibers, COX-negative staining fibers, or abnormal accumulations of lipid. 3MGA levels were considered abnormal if they were greater than 2 standard deviations from the mean values in our laboratory. Results: 7/8 patients with myopathy on biopsy had abnormal 3MGA 0/3 patients with no myopathy on biopsy had abnormal 3MGA 0/6 patients on statins without muscle complaints had abnormal 3MGA Conclusion: In a small series, using abnormal muscle biopsies as the gold standard, 3MGA appears 87.5 % sensitive and 100% specific in confirming statin associated myopathy with normal CK. P221 A Novel Gas Chromatograph/Mass Spectrometric Method (GC/MS) to Measure Vascular Smooth Muscle Cell Proliferation, In Vivo, Using 2H2O Alice Chu, Eric T Ordonez, Marc K Hellerstein. University of California at Berkeley, Berkeley, CA VSMC proliferation plays a pathogenic role in atherosclerosis and other hyperplastic diseases of the vessel wall. We have developed a method to measure VSMC turnover using 2 H 2O (heavy water) to label the deoxyribose (dR) moiety of DNA. The method is based on the exchange of deuterium into dR in the formation of deoxyribonucleosides (dN) used for DNA replication. Mice are injected with 100% 2 H 2O to reach a body water enrichment of 2% and maintained on 4% 2 H2O in drinking water for three weeks. Upon sacrifice, the aorta and bone marrow (BM) are removed. Aorta is subjected to collagenase treatment to remove adventitial and endothelial layers. DNA is extracted and enzymatically hydrolyzed to dN. Deoxyadenosine is isolated, mild acid-hydrolyzed to dN to remove adenine, and derivitized to dR-pentane tetraacetate for analysis by GC/MS (m/z 245,246). Calculation of f(%new cells)(Enrichment of VSMC/ Enrichment of BM)*100 and k (%new cells/day){-ln(1-f)/t}*100. We initially tested the method in young (age 3–6 weeks) and adult (age 15–21 weeks) C57Bl/6J mice. Significantly higher f (5.73.5% vs 2.41.5%) and k (0.270.16% vs 0.120.08%) were observed in the 9 week vs 21 week old mice (P0.05). A delabeling study was also conducted. Mice were labeled for 10 weeks then switched to non-labeled drinking water and sacrificed every 2 weeks. VSMC enrichments remained nearly constant, confirming slow turnover. VSMC proliferation was compared in apoE-/- and C57Bl/6J mice fed low-fat or high-fat diet from age 5 weeks. AFter 9 weeks of diet, f of apoE-/- high-fat increased and remained significantly higher than all other groups (263% vs 1–8%, P0.01). The increased VSMC proliferation preceded and ultimately correlated with histologic changes in the aortic wall. In conclusion, the method allows the measurement of VSMC turnover and proliferation in vivo, confirms the normally slow turnover rate of VSMC in rodents, and demonstrates early changes during the progression of atherosclerosis; thereby representing a potentially sensitive measure of atherogenesis. P222 Hyperhomocysteinemia Induces Endothelial Apoptosis: Mechanism and Implications for Atherogenesis Ganesh Raveendran, Kalpna Gupta, Anna Solevey, Liming Cheng, Robert Hebbel. University of Minnesota, Minneapolis, MN Background: Hyperhomocysteinemia is an independent risk factor for cardiovascular mortality. Mechanism of action of hyperhomocysteinemia leading to increased cardiovascular morbidity and mortality is unknown. Endothelial cell (EC) injury, and perhaps apoptosis, are the key events in the pathogenesis of atherosclerosis and represent an initial step in atherogenesis. Methods: We hypothesized that endothelial cells from patient with homozygote state for cystathionine--synthase (CBS) deficiency will have higher apoptosis and higher expression of adhesion molecule and tissue factor. BOECs were cultured from blood of one homozygote with CBS deficiency and two normal controls. BOECs were treated with regular EC medium, serum free medium and escalating concentration of methionine and homocysteine (Hcy). Apoptosis was estimated by ELISA using optical density measurements and by tunnel assay after 48 hours. Each experiment was performed in duplicate with 8 samples for each test. Results were analyzed using independent Students T test. Results: Our preliminary results by ELISA show BOECs from CBS deficient patient had statistically significant difference in apoptosis compared to the normal controls. Serum free medium induced apoptosis in both group but did have any significant difference between the groups. Similar comparable results were observed in tunnel assay. Methionine loading in the culture medium did not have an effect on the measured Hcy level from the supernatant. Poster Presentations a-39 effects, Ang II is a potent stimulus for adrenal production of aldosterone, which may also cause myocardial and vascular injuries. In other studies of ApoE-deficient mice we found that mineralocorticoid excess produced by infusion of deoxycorticosterone acetate (DOCA-50 mg pellet implanted for 28 days starting at 8 weeks age) and salt loading increased lesion area of the descending aorta from 0.40.1% in controls to 549% in treated animals (p0.0001), while having little effect at the aortic root. Objectives: To determine the role of mineralocorticoid excess in mediating the effects of Ang II on atherogenesis we measured plasma aldosterone levels in Ang II-infused mice and then reproduced these levels by direct infusion of aldosterone via osmotic minipump. Methods: Plasma aldosterone levels measured 4 weeks after Ang II infusion via osmotic minipump (1,000 ng/kg/min) increased from 185 to19837 ng/dl (n 4, p0.003). Osmotic minipumps were implanted to deliver aldosterone to achieve similar levels. The final infusion rates were 200 and 400 /kg/day. Findings: After 28 days infusion, these infusion rates produced plasma aldosterone levels of 13513 and 29857 (n6 for each group), which spanned the plasma levels observed in the Ang II-infused mice. Systolic blood pressure, measured by tail cuff increased significantly in both groups by 13 mmHg compared to sham operated controls, as did kidney weight/body weight ratio. However, in both aldosterone infusion groups there was no increase in atherosclerosis at either the aortic root or in the descending aorta. Conclusion: These studies suggest that mineralocorticoid excess only increases atherosclerosis under the pharmacological conditions of DOCA/salt treatment. If aldosterone is involved in mediating atherosclerosis in response to Ang II, then plasma Ang II levels must also be elevated for aldosterone to exert its effects. Alterations in Apo A-I Conformation Accompanying the Conversion of Discoidal to Spherical High Density Lipoproteins Hui-Hua Li, Douglas S Lyles, Michael J Thomas, Wei Pan, Eric Alexander, Michael Samuel, Mary G Sorci-Thomas. Wake Forest University School of Medicine, Winston-Salem, NC P224 Apolipoprotein A-I (apo A-I) readily binds and organizes phospholipid bilayers to form discoidal HDL particles. In the lipid-bound form, apo A-I activates lecithin:cholesterol acyltransferase (LCAT), which generates cholesterol ester (CE) from free cholesterol (FC). As esterification proceeds discoidal HDL transforms into a spherical HDL with accumulation of cholesterol esters within the hydrophobic core of the bilayer. A typical 96Ã. . . diameter discoidal HDL particle contains two molecules of apo A-I and is oriented in an antiparallel “belt” conformation surrounding a phospholipid bilayer. However, the conformational changes in apo A-I structure that take place as the discoidal HDL conforms to the presence of a hydrophobic core are currently unknown. In order to characterize the changes in apo A-I structure as it adapts to a sphere, we measured the inter-molecular distance between apo A-I molecules using fluorescence resonance energy transfer (FRET). Selected domains on each of 2 lipid-bound apo A-I molecules were studied in both the discoidal and the spherical HDL form. These data showed that as discoidal particles were converted to spherical HDL through the action of LCAT, compositional changes included a 35% decrease in phospholipid, 70% decrease in FC, and a 94% increase in CE mass. In addition, after the conversion, spherical HDL particles were found to have obtained a third molecule of apo A-I, as determined by crosslinking analysis. FRET analysis showed that the inter-molecular distance between domains corresponding to repeat 5 (amino acids 121–142) showed very little change, while the domain corresponding to repeat 6 (amino acids 143–164) showed a much larger inter-molecular shift as CE accumulated. These data suggest that the favored “belt conformation” found on discoidal HDL is significantly altered after the particle accumulates CE in the core. P225 Selective Cyclooxygenase-2 Inhibition Decreases Monocyte Chemoattractant Protein-1 Expression and Neointimal Hyperplasia in the Rabbit Atherosclerotic Balloon Injury Model Kai Wang, Zhongmin Zhou, Khaldoun Tarakji, A Michael Lincoff, Eric J Topol, Marc S Penn. The Cleveland Clinic Foundation, Cleveland, OH Angiotensin II Infusion Increases Plasma Aldosterone Levels and P223 The inflammation in response to vascular injury is becoming increasingly recognized as a potential contributor to restenosis. Cyclooxygenase-2 (COX-2) has been shown to be involved in the proinflammatory response of vascular tissue, potentially through regulation of monocyte chemoattractant protein-1 expression (MCP-1). We hypothesized that specific COX-2 antagonism would lead to decreased MCP-1 expression, inhibiting inflammatory response following vascular injury, and ultimately reducing neointimal formation after vascular injury. Methods and Results: Bilateral focal femoral artery lesions were induced by air desiccation in New Zealand White rabbits followed by high cholesterol diet feeding for 28 days. Balloon injury was then induced at the pre-injured vessel segments by three balloon inflations. Initial studies using immunostaining (n4 animals) showed that uninjured vessel segments stained positive only for COX-1 but not for COX-2. Injured vessel segments showed, in addition to COX-1, significant positive staining for COX-2 in both endothelium and macrophages, demonstrating upregulation of expression of this pro-inflammatory enzyme in response to injury. In the efficacy study, celecoxib (75mg/kg/day) was administered orally beginning 3 hours before balloon injury on Accelerates Aortic Atherosclerosis in ApoE-Deficient Mice, but Aldosterone day 28 and then daily for 21 days. MCP-1 expression was quantified in arterial extracts 4 days Infusion Alone Has No Effect after balloon injury by western blot. Morphometric analysis and immunostaining for macrophages was performed 21 days after balloon injury. Celecoxib treatment significantly decreased Ying Zhou, Rong Chen, Yuexian Shi, Lufei Hu, Jean E Sealey, John H Laragh, Hayes M MCP-1 activity (5.3Â1.9 VS 29.0Â4.6 arbitrary units, p0.01), and neointimal hyperplasia Dansky, Daniel F Catanzaro. Weill Medical College, Cornell University, New York, NY; Mount by more than 30% (0.49Â0.20 vs 0.70Â0.35 mm2, p0.05), accompanied by reduced Sinai School of Medicine, New York, NY macrophage infiltration. Conclusions: Selective antagonism of COX-2 decreases the inflammatory response and intimal hyperplasia following vascular injury, possibly due to the early Background: Recent studies show that Ang II infusion increases atherosclerosis at both the inhibition of MCP-1 expression, implying a pivotal role of inflammation in the pathogenesis of aortic root and in the descending aorta of ApoE-deficient Downloaded mice. In addition from tohttp://atvb.ahajournals.org/ its vasoconstrictor restenosis. by guest on April 4, 2013
a-40 Arterioscler Thromb Vasc Biol. Vol 22, No 5 P226 Small, Dense LDL, Unstable Carotid Plaque and Cerebrovascular Events in Patients Undergoing Carotid Endarterectomy Alberto Zambon, Elisabetta Faggin, Sandra Bertocco, Nicola Vitturi, Massimo Puato, Gaetano Crepaldi, Paolo Pauletto. University of Padova School of Medicine, Padova, Italy Low-density lipoprotein (LDL) size and density contribute to LDL particle atherogenicity. The presence of small, dense LDL particles is associated with: 1) a three to six fold increased risk of coronary artery disease, and 2) with an increased carotid intima-media thickness, a marker of early atherosclerosis, in normolipidemic subjects. We investigated the possible association between LDL density, cell composition of carotid plaques (unstable plaques) and the occurrence of ischemic cerebrovascular events (ICVE). Sixty-eight patients undergoing carotid endarterectomy, with a carotid stenosis greater than 70% of the lumen, were studied. Plasma LDL density was evaluated by density gradient ultracentrifugation. Endarterectomy specimens were examined by immunocytochemistry using the following monoclonal antibodies: i) the SM-E7 identifying smooth muscle cells (SMCs), ii) the anti-macrophage HAM 56, and iii) the anti-lymphocyte CD45R0. Fifty patients had at least one episode of ICVE prior the endarterectomy, while 18 had no ICVE. Mean blood pressure, gender, BMI, total, LDL and HDL cholesterol, TG, Lp(a), apo AI and apo B levels, smoke habits, glucose, fibrinogen, plasminogen and homocysteine levels, were not significantly different in those with previous ICVE as compared with patients without ICVE. Patients with previous ICVE had significantly denser LDL particles (p0.01) and more macrophages (p0.01) than those without ICVE. LDL density was significanlty associated with both the number of macrophages (r 0.59, p0.01) and SMCs (r -0.50, p0.01) in the plaque. In a multivariate analysis including clinical and lipid variables, LDL density was the only parameter significantly predicting the number of macrophages in the plaque (p0.01). In a logistic regression analysis the number of macrophages was the strongest predictor of ICVE (p0.01). The presence of dense LDL particles is associated with an abundance of inflammatory cells in the carotid plaque (unstable plaque) and excess prevalence of ICVE in subjects with carotid stenosis. P227 Delineation of the Role of Pre 1-HDL in Cholesterol Efflux Using Isolated Pre 1-HDL Dmitri Sviridov, Osamu Miyazaki, Kally Theodore, Anh Hoang, Isamu Fukamachi, Paul Nestel. Baker Medical Research Institute, Melbourne, Australia; Diagnostic Research Laboratories, Daiichi Pure Chemicals Ltd, Tokyo, Japan The role of pre 1-HDL in cholesterol efflux was investigated by separating human plasma into purified pre 1-HDL and pre 1-HDL -deficient plasma using a monoclonal antibody specifically reacting with pre 1-HDL. When compared to whole plasma, pre 1-HDL-deficient plasma was equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human macrophage cells. When added at the same apoA-I concentration pre 1-HDL was less effective than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective with THP-1 cells. However, pre 1-HDL-deficient plasma reconstituted with 16% pre 1-HDL was more active than whole plasma demonstrating that pre 1-HDL does promote cholesterol efflux actively. The amount of cellular cholesterol present in re-isolated pre 1-HDL was 1.5–2 -fold greater following incubation of cells with whole plasma, than with pre 1-HDL-deficient plasma or plasma treated with the antibody. Time-course efflux experiments showed that isolated pre 1-HDL was not significantly different to whole plasma in promoting efflux from “fast” cholesterol pool, but less active in recruiting cholesterol from a “slow” pool. Anti pre 1-HDL antibody did not inhibit cholesterol efflux to whole plasma, pre 1-HDL-deficient plasma or isolated pre 1-HDL and the epitope of this antibody is located between residues 140–210, i.e. some distance from lipid-binding domains of apolipoprotein A-I. We conclude that whereas pre 1-HDL are capable of taking up cellular cholesterol, their presence in plasma is not essential for cholesterol efflux, at least in vitro. Instead pre 1-HDL may be the first product of apoA-I lipidation during formation of HDL, but might not play a major role in transferring cellular cholesterol to HDL Cilostazol Effects on Neointimal Growth in the Carotid Artery of Rats Following Balloon Injury Dadong Li, Laurine Bow, Xin Yang, Charles Nightingale, Ronald Langner. Hartford Hospital, Hartford, CT; University of Connecticut, Storrs, CT P228 Restenosis of blood vessels following percutaneous coronary angioplasty (PTCA) is a significant medical problem. Platelet aggregation and the subsequent release of platelet cytokines is thought to play an important role in promoting blood vessel restenosis. The purpose of this study was to determine the ability of cilostazol, a phosphodiesterase type III inhibitor, to inhibit platelet aggregation and to inhibit restenosis in rats. The endothelial lining of the left carotid artery of 16 male Sprague-Dawley rats was injured by inflating a balloon catheter and moving it back and forth four times. The 16 rats were randomly divided into two groups. One group was treated orally with cilostazol, 135mg/kg, twice a day while the other group was treated with vehicle. Drug and vehicle administration began one day before balloon-injury and was continued for 15 days. Cilostazol significantly inhibited platelet aggregation after 7 days of treatment and remained constant through the remainder of the 15 days treatment period. The extent of neointimal growth in the carotid arteries was estimated by measuring the areas of intima and media with a computerized morphometric analyzer. The balloon-injured carotid arteries from vehicle treated rats had a significantly thickened intimal layer which was characterized by increased proliferation of smooth muscle cells and deposition of connective tissue compared to its uninjured right carotid artery. In the cilostazol treated rats the intimal layer of the injured carotid artery was also significantly thicker compared to the uninjured right carotid artery, however the degree of intimal proliferation in these animals was significantly decreased compared to that of injured arteries from vehicle treated rats. The data suggests that treatment with cilostazol may have a beneficial effect in retarding the extent of restenosis in blood vessels following PCTA. Downloaded from http://atvb.ahajournals.org/ Modulation of Arterial Healing in Heterozygous Watanabe Rabbits by Aspirin and Verapamil P229 Kenneth J Woodside, Howard H Hayashi, Linda Talken, John A Daller, Charlie Luo, Glenn C Hunter. University of Texas Medical Branch, Galveston, TX; University of California at Davis, Sacramento, CA There is increasing evidence that modulating the components of complex atherosclerotic plaque may reduce the frequency of symptoms and delay progression of disease. This study evaluates the effects of aspirin (ASA) and verapamil on the nonlipid components of complex plaque induced in heterozygous Watanabe Heritable Hyperlipidemic (HWHHL) rabbits by balloon catheter injury. METHODS: Balloon catheter aortic injury was induced in 27 HWHHL rabbits: 9 control, 9 received 5 mg/kg of aspirin, and 9 receiving 0.15 g/kg verapamil orally daily. Serum and tissue cholestrol and drugs levels were assayed. Three animals each were sacrificed at 1, 3, and 6 months. The aortas were examined histologically for evidence of neointimal hyperplasia (NFH), thrombus formation, and calcification. RESULTS: Two animals died during the study period. Lipid alterations and NFH at the later timepoints are shown in the Table. CONCLUSIONS: Aortic balloon catheter injury in this model can reproduce all the components of complex atherosclerotic plaque and allows pharmacologic modulation of each specific component. Although verapamil appeared to be more advantageous than ASA in altering NFH, thrombus, and calcification, it seems likely that multiple agent therapy may be required in order to reduce plaque instability. Effects of Preprandial Ethanol on Cholesteryl Ester Transfer in Normotriglyceridemic and Hypertriglyceridemic Subjects John W Gaubatz, Lynne W Scott, Kay T Kimball, Lisa Wu, Christie M Ballantyne, Henry J Pownall. Baylor College of Medicine and The Methodist Hospital, Houston, TX P230 The effects of standard acute doses of alcohol, saturated fat (SF), and alcohol plus SF on plasma cholesteryl ester transfer activity (CETA) and concentrations of cholesterol, nonesterified fatty acids (NEFA), and triglyceride (TG) were determined in 9 normotriglyceridemic (NTG) and 4 moderately hypertriglyceridemic (HTG) subjects (respective fasting TG 1.19 0.19 mmol/L and 4.93 1.20 mmol/L, mean SEM). Over the 10 hours following the ingestion of alcohol, CETA, cholesterol, and TG values did not significantly change in either group, whereas NEFA transiently decreased recovering to 100% and 150% of the baseline value at 10 hours in the NTG and HTG groups. In both groups, the fat challenge was followed by an increase in CETA, NEFA, and TG; cholesterol was not significantly affected. Alcohol plus SF did not affect cholesterol but, compared with SF alone, increased CETA and the magnitude of lipemia and suppressed the increase in NEFA in both groups. The fractional increases in CETA and TG with addition of alcohol were positively correlated in both the NTG (r 2 0.91) and HTG (r 2 0.80) groups; the increase in CETA with TG was 3.5 times greater in HTG than in NTG. However, a single line of regression was obtained for the correlation of CETA and TG (r 2 0.62; P 0.0001). This suggests that CETA is a direct function of TG concentration in both NTG and moderately HTG subjects, and that intestinally derived lipoproteins are the major acceptors of HDL-CE, particularly in subjects consuming fat with alcohol. Thus, CETA may be involved in the cardioprotection conferred by regular, moderate consumption of alcohol. This protection could occur through the increased diversion of HDL cholesterol to intestinally derived lipoproteins, which are destined for hepatic uptake. P231 Adventitia-Dependent Intima Hyperplasia and Vasoconstriction of Carotid Arteries in Rabbits on Cholesterol-Rich Diet Zhiming Zhu, Huaming Mu. Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing, China Background: Adventitial cells may play an important role for the neointima formation and modulation of vascular smooth muscle cell phenotype. The objective of the present study was to test whether adventitial cells in hypercholesterolemic rabbits on a cholesterol-rich diet directly affect neointima formation and phenotype of carotid artery. Methods: In 8 rabbits adventitia was excised from one carotid artery before receiving a cholesterol-rich (2%) diet for 6 weeks. Eight rabbits receiving standard chow were sham-operated. After 1 week and 6 weeks carotid arteries were isolated for histological examination, and vasoconstriction was measured in carotid rings. Results: After 1 week rabbits on a cholesterol-rich diet showed a severe hyperplastic intimal lesion after removal of the adventitia compared to sham-operated rabbits. Only collagen type I were observed in carotid arteries of rabbits on a cholesterol-rich diet without adventita, whereas collagen type I and type III could be observed in sham-operated rabbits. KCl-induced vasoconstriction was significantly reduced (1.57/-0.16 g vs 0.57/- 0.08 g, p0.01), whereas acetylcholine (Ach)-induced relaxation was significantly enhanced in carotid arteries of rabbits on a cholesterol-rich diet without adventitia compared to shamoperated rabbits by guest ( 0.14/-0.09 on April g4, vs 2013 0.25/-0.08 g, p0.05). After 6 weeks rabbits on a
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