bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of Vitamins C and E Alters the Response to Coronary Balloon Injury in the Pig
Authors: Nunes Gilberto L. , Sgoutas Demetrios S. , Redden Robert A. , Sigman Steven R. , Gravanis Michael B. , King Spencer B. , Berk Bradford C.
Published: June 2011

Abstract
Restenosis is the major limitation of the long-term success of percutaneous transluminal coronary angioplasty. The process of restenosis involves repair of vascular injury and remodeling of vessel architecture. Therapeutic interventions that improve vascular function may therefore be beneficial in the treatment of restenosis. Antioxidants such as probucol and vitamins C and E have proved effective in improving endothelial function in hypercholesterolemia, inhibiting lipid accumulation in animal models of atherosclerosis, and decreasing cardiovascular mortality in humans. Forty-two female domestic swine were divided into four study groups: control (n=12); vitamin C (500 mg/d, group C, n=9); vitamin E (1000 U/d, group E, n=10); and vitamins C and E (500 mg/d + 1000 U/d, group C + E, n=11) before oversized balloon injury of the left anterior descending and circumflex coronary arteries. Vitamins were administered 7 days before balloon injury and continued until the swine were killed 14 days after injury. Significant differences in morphometric parameters were present only in group C + E, with increases in vessel and lumen area in the segment with maximal injury. Although there was no decrease in intima area or in maximal intima thickness, the ratio of intima area to vessel area was significantly reduced, consistent with a positive effect in group C + E. Graphic analysis of the relationship between initial vessel injury (using internal elastic lamina fracture length/lumen perimeter) and vessel response to injury (using intima area/vessel area) for all segments showed improved indices for group C + E (
P
<.005). The beneficial effect of vitamins correlated with changes in lipid redox state. Low-density lipoprotein (LDL) thiobarbituric acid–reactive substances showed an ≈70% decrease in all treatment groups, and the lag phase for LDL-conjugated diene formation was significantly increased, with group C + E>group E>group C. The combination of vitamins C and E improved vascular response to injury because of an apparent beneficial effect on vascular remodeling. The fact that the combination of vitamins C + E was better than vitamin E or vitamin C alone is consistent with the ability of vitamin C to improve the antioxidant effect of vitamin E, suggesting that the improved vessel response was due to a change in redox state. This study suggests an important role for oxygen radicals in the vascular response to injury and suggests that vascular remodeling and intimal proliferation are important to the restenotic process.
Expression of ICAM-1 and VCAM-1 and Monocyte Adherence in Arteries Exposed to Altered Shear Stress
Authors: Walpola Piyal L. , Gotlieb Avrum I. , Cybulsky Myron I. , Langille B. Lowell
Published: June 2011

Abstract
Local shear stresses generated by blood flow exert direct mechanical effects on adhesion of circulating leukocytes to vascular endothelium, but their effects on expression of endothelial-leukocyte adhesion molecules have not been determined. Shear stress in rabbit carotid arteries was increased by 170% or decreased by 73% in 5 days by surgical manipulations. En face immunofluorescence staining with the monoclonal antibody Rb1/9 revealed that vascular cell adhesion molecule–1 (VCAM-1) expression was greatly increased under low shear stress, but the distribution of staining was patchy. Thus, 71.4±7.8% of fields were VCAM-1 positive versus 2.4±0.47% of fields in control arteries. Frequently, large regions showed consistent but heterogeneous staining. Occasionally, small islands of cells were labeled intensely. Monocytes, detected by use of the monocyte-specific antibody HAM 56, adhered to endothelium under low shear stress; 64.5±8.2% of the monocytes colocalized with detectable VCAM-1, although many (83.2±2.8%) VCAM-1–positive regions were devoid of monocytes. VCAM-1 expression also increased significantly but to a lesser extent when shear stress was approximately doubled. Thus, 8.7±1.5% of fields were VCAM-1 positive under high shear versus 2.5±0.87% under normal shear stress. No monocytes were detected at high shear stress. At normal shear stresses, intercellular adhesion molecule–1 (ICAM-1), detected by use of the monoclonal antibody Rb2/3, was extensively distributed; thus, 53.5±5.5% of fields contained ICAM-1–positive cells. The junctional regions of the cells were heavily stained. Experimental increases in shear stress significantly upregulated ICAM-1 expression (88.3±2.0% of fields were ICAM-1 positive; P<.05). Staining was again concentrated in the cell junctional regions. Reduced shear stress suppressed ICAM-1 expression (16.6±10.3% of fields were positive), and ICAM-1 was more commonly, but not exclusively, distributed diffusely. Junctional ICAM-1 may participate in endothelial cell-cell adhesion; alternatively, a pool of ICAM-1 concentrated between cell junctions may be inaccessible to circulating leukocytes until endothelial cell activation presents the molecule to the vessel lumen.
Augmented Urokinase Receptor Expression in Atheroma
Authors: Noda-Heiny Hiroko , Daugherty Alan , Sobel Burton E.
Published: June 2011

Abstract
Smooth muscle cell proliferation and migration into neointima are hallmarks of atherogenesis. However, mechanisms responsible have not yet been fully elucidated. One potential mediator of both smooth muscle cell proliferation and migration is activation of plasminogen by activators bound to receptors on cells within the vessel wall. To determine whether vascular smooth muscle cells within atheroma express the receptor for urokinase-type plasminogen activator (uPA-R), we characterized atheroma in cholesterol-fed New Zealand White rabbits and human subjects by immunostaining. Intense immunostaining of uPA-R was observed throughout the neointima in both rabbit and human atherosclerotic lesions with the use of a monoclonal antibody to uPA-R. uPA-R was not detectable in normal arterial tissues. uPA-R was localized to macrophages and neointimal smooth muscle cells identified by immunostaining in serial sections. Furthermore, uPA-R protein in extracts from atheroma was present in at least a ninefold greater quantity compared with extracts from normal vessels, as shown by Western blotting. Expression of uPA-R mRNA in migrating vascular smooth muscle cells did not increase significantly. Thus, altered posttranscriptional regulation may be contributing to the increased uPA-R. In vitro, antibodies to uPA-R delayed the migration of cultured vascular smooth muscle cells. Our results suggest that increased cell-surface uPA-R contributes to pericellular proteolysis and consequently increased neointimalization secondary to increased vascular smooth muscle cell migration in atheroma.
A Polymorphism of the Paraoxonase Gene Associated With Variation in Plasma Lipoproteins in a Genetic Isolate
Authors: Hegele Robert A. , Brunt J. Howard , Connelly Philip W.
Published: June 2011

AbstractThe Hutterite Brethren are a genetic isolate characterized by high indices of relatedness and a communal agrarian lifestyle. We hypothesized that variation of the paraoxonase (PON) gene that underlies the interindividual variation in plasma PON activity would be associated with variation in fasting plasma lipoprotein variables in this group. In 793 Hutterites, we measured plasma lipids, lipoproteins, and apolipoproteins and analyzed DNA for genotypes of the protein polymorphism at amino acid residue 192 of PON. We observed that genotypes of PON were significantly associated with variation in plasma concentrations of total, HDL, non-HDL, and LDL cholesterol, total triglycerides, and apolipoprotein (apo) B. Homozygotes for the low-activity variant of PON had significantly lower levels of plasma apoB-related biochemical variables than heterozygotes and homozygotes for the high-activity variant of PON. Homozygotes for the low-activity variant of PON also had significantly lower ratios of total cholesterol/HDL cholesterol, LDL cholesterol/HDL cholesterol, and apoB/apoA-I than heterozygotes and homozygotes for the high-activity variant of PON. We found no evidence for a gene-gender interaction for any plasma lipoprotein variable. The PON polymorphism accounted for about 1% of the variation in total cholesterol and related lipoprotein traits in the Hutterites. These observations suggest that PON is a significant genetic determinant of plasma lipoprotein levels.
Oxidized LDL Induces Enhanced Antibody Formation and MHC Class II–Dependent IFN-γ Production in Lymphocytes From Healthy Individuals
Authors: Huang Yi Hui , Rönnelid Johan , Frostegård Johan
Published: June 2011

Abstract
The early stages of atherosclerosis are characterized by penetration into the arterial intima by both T lymphocytes and monocytes. Some of these T lymphocytes show signs of activation, though the mechanisms by which they become activated are not known. The monocytes develop into macrophages and subsequently into foam cells filled with oxidized LDL (oxLDL)-derived lipids. OxLDL has been found to exert several proinflammatory effects, including enhanced adhesiveness of endothelial cells and monocytes, chemotaxis of monocytes and T cells, and T-cell activation. The enzyme-linked immunospot (ELISPOT) assay has been shown to be a sensitive method for detection of single cells secreting antibodies or cytokines. Here we have used this method to characterize the T-cell cytokine secretion pattern after exposure to oxLDL in vitro. In peripheral blood mononuclear cells from healthy donors (n=27), a significantly enhanced number of INF-γ–producing cells was detected by ELISPOT (
P
<.001) after stimulation with 5 μg/mL oxLDL. In contrast, production of interleukin-4 was not significantly enhanced after stimulation with oxLDL. OxLDL-induced IFN-γ secretion and T-cell proliferation were completely inhibited by major histocompatibility complex (MHC) class II antibodies. Furthermore, oxLDL was found to enhance the antibody secretion, indicating B-cell activation. Our results indicate that oxLDL activates T cells by an MHC class II–dependent mechanism. In healthy individuals, oxLDL induces IFN-γ, which is produced by T helper type 1–like cells. These findings demonstrate that oxLDL induces a cell-dependent immune reaction, which may play an important role in the development of atherosclerosis.
Inhibition of Lecithin-Cholesterol Acyltransferase and Modification of HDL Apolipoproteins by Aldehydes
Authors: McCall Mark R. , Tang Jean Y. , Bielicki John K. , Forte Trudy M.
Published: June 2011

Abstract
Experimental evidence suggests that aldehydes generated as a consequence of lipid peroxidation may be involved in the pathogenesis of atherosclerosis. It is well documented that aldehydes modify LDL; however, less is known concerning the effects of aldehydes on other plasma and interstitial fluid components. In the present study, we investigated the effects of five physiologically relevant aldehydes (acetaldehyde, acrolein, hexanal, 4-hydroxynonenal [HNE], and malondialdehyde [MDA]) on two key constituents of the antiatherogenic reverse cholesterol transport pathway, lecithin-cholesterol acyltransferase (LCAT) and HDL. Human plasma was incubated for 3 hours at 37°C with each one of the five aldehydes at concentrations ranging from 0.16 to 84 mmol/L. Dose-dependent decreases in LCAT activity were observed. The short-chain (acrolein) and long-chain (HNE) α,β-unsaturated aldehydes were the most effective LCAT inhibitors. Micromolar concentrations of these unsaturated aldehydes resulted in significant reductions in plasma LCAT activity. The short- and longer-chain saturated aldehydes acetaldehyde and hexanal and the dialdehyde MDA were considerably less effective at inhibiting LCAT than were acrolein and HNE. In addition to inhibiting LCAT, aldehydes increased HDL electrophoretic mobility and cross-linked HDL apolipoproteins. Cross-linking of apolipoproteins A-I and A-II required higher aldehyde concentrations than inhibition of LCAT. The α,β-unsaturated aldehydes acrolein and HNE were fourfold to eightfold more effective cross-linkers of apolipoproteins A-I and A-II than the other aldehydes studied. These data suggest that products of lipid peroxidation, especially unsaturated aldehydes, may interfere with normal HDL cholesterol transport by inhibiting LCAT and modifying HDL apolipoproteins.
Angiotensin II Increases cGMP Content Via Endothelial Angiotensin II AT1 Subtype Receptors in the Rat Carotid Artery
Authors: Caputo Lidia , Benessiano Joëlle , Boulanger Chantal M. , Lévy Bernard I.
Published: June 2011

Abstract
Angiotensin II (Ang II) has been reported to modulate cGMP formation in various types of cells. To acquire direct information on the intracellular transduction involved in this mechanism, we tested the effects of Ang II on vascular tone and on cGMP content of in vitro isolated carotid arteries from 12-week-old Wistar-Kyoto rats. Segments of carotid artery 20 mm long (n=8 for each group) maintained at a transmural pressure of 100 mm Hg were immersed in a bath (38°C) containing oxygenated Tyrode’s solution. At the end of each experiment, the vessel diameter was measured, and the wall cGMP content was determined by enzyme immunoassay. Under basal conditions, mean diameter was 968±19 μm, and mean cGMP carotid artery content was 38.9±3.5 fmol/mg tissue. Incubation for 20 minutes with Ang II (10
−5
mol/L) significantly increased cGMP wall content, twofold above the basal content (
P
<.01), and constricted the vessel (60±2.2% of the control diameter,
P
<.001). After preincubation with a nonselective antagonist of Ang II receptors, saralasin ([Sar
1
,Val
5
,Ala
8
]Ang II, 5×10
−5
mol/L), or with a specific antagonist of Ang II AT1 receptor subtype, losartan (5×10
−5
mol/L), carotid diameter and cGMP content were no longer affected by Ang II. Exposure of carotid arteries to a specific antagonist of Ang II AT2 receptor, PD 123319 (10
−7
mol/L), modified neither Ang II–induced diameter decrease nor cGMP content increase. Constriction of the vessel with KCl (26±3%,
P
<.001) did not modify the basal cGMP wall content. Endothelium removal or incubation with
N
G
-nitro-
l
-arginine methyl ester (10
−3
mol/L) reduced the cGMP content (22±9%,
P
<.05 and 20±11%,
P
<.05, respectively); Ang II further decreased the diameter (
P
<.001) but no longer increased the cGMP content under these experimental conditions. The present study shows that Ang II constricts the carotid artery and increases cGMP level specifically via the Ang II AT1 receptor subtype in in vitro intact rat carotid artery. The mechanism underlying this increase in cGMP is thought to be mediated through endothelial NO synthase stimulation by Ang II.
Retinoids Stimulate Fibrinogen Production Both In Vitro (Hepatocytes) and In Vivo
Authors: Nicodeme Edwige , Nicaud Michael , Issandou Marc
Published: June 2011

Abstract
The in vitro effects of retinoids on fibrinogen synthesis were investigated in HepG2 cells and primary human hepatocytes. In vivo effects were studied in the rat. In HepG2 cells, maximal stimulation (twofold) of fibrinogen secretion was obtained when cells were incubated in the presence of 1 μmol/L all-
trans
retinoic acid (T-RA) for 24 hours. A comparable increase was observed for both de novo fibrinogen synthesis and fibrinogen β chain mRNA level. In primary cultures of human hepatocytes, treatment with 1 μmol/L T-RA for 72 hours also gave a twofold increase in fibrinogen production. Furthermore, rats treated for 6 days with 100 mg · kg
−1
· d
−1
T-RA presented increased fibrinogen plasma levels (110%). A selective retinoic X receptor (RXR) agonist, 4-[1-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid (3-methyl TTNEB), as well as 9-
cis
retinoic acid, a natural RXR ligand, mimicked the effects of T-RA on fibrinogen synthesis in vitro at lower concentrations. In contrast, a selective retinoic A receptor α (RARα) agonist was a poor activator. The ED
50
of the different retinoids on fibrinogen secretion by HepG2 cells was 25 nmol/L for T-RA, 4 nmol/L for 9-
cis
retinoic acid, 11 nmol/L for the synthetic RXR agonist, and >500 nmol/L for the RARα agonist. However, incubation of HepG2 cells with RXR agonist together with RARα agonist resulted in a further increase in fibrinogen production. The secretion of two other acute-phase proteins, α-antichymotrypsin and caeruloplasmin, was also stimulated by retinoids in HepG2 cells but by a different regulatory mechanism. We conclude that activation of RXR by a specific ligand upregulates fibrinogen production by hepatocytes. Elevated levels of fibrinogen in rats treated with T-RA indicate that retinoids may be involved in the physiological regulation of fibrinogen, with a key role for RXR.
Heightened Thrombin Formation but Normal Plasma Levels of Activated Factor VII in Patients With Acute Coronary Syndromes
Authors: Merlini Piera Angelica , Ardissino Diego , Oltrona Luigi , Broccolino Marco , Coppola Raffaella , Mannucci Pier Mannuccio
Published: June 2011

AbstractPlaque rupture with the exposure of a tissue factor–rich procoagulant surface is considered the common pathogenetic mechanism of unstable angina and myocardial infarction. Activated factor VII, the key enzyme for initiating blood coagulation under resting conditions, is increased in pathological situations associated with tissue factor exposure. We measured the plasma levels of activated factor VII and studied their relation with signs of coagulation enzyme activity in patients with acute coronary syndromes. The plasma levels of activated factor VII, prothrombin fragment 1+2, and fibrinopeptide A were measured on admission in consecutive patients presenting with acute myocardial infarction (n=28), unstable angina (n=32), and stable angina (n=17) and in age- and sex-matched healthy individuals (n=33). Plasma determinations of the same markers were also repeated at 15 days and 3 and 6 months. On admission, the patients with unstable angina or myocardial infarction had significantly higher plasma levels of prothrombin fragment 1+2 (P<.0001) and fibrinopeptide A (P<.0001) than those with stable angina or healthy individuals, whereas no differences were detected in the plasma levels of activated factor VII. During follow-up there was a significant decrease in the plasma levels of fibrinopeptide A both in patients with unstable angina (P<.001) and in those with myocardial infarction (P<.001), whereas no changes in plasma prothrombin fragment 1+2 or activated factor VII levels were observed. Hence, in the acute and chronic phases of myocardial infarction and unstable angina, heightened coagulation enzyme activity is not accompanied by an increase in activated factor VII.
Lipoproteins Containing Apolipoprotein A-IV but Not Apolipoprotein A-I Take Up and Esterify Cell-Derived Cholesterol in Plasma
Authors: von Eckardstein Arnold , Huang Yadong , Wu Shili , Sarmadi Ahmad Saadat , Schwarz Sigrid , Steinmetz Armin , Assmann Gerd
Published: June 2011

Abstract
Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) identifies distinct apoA-I– or apoE-containing subclasses of high-density lipoproteins (HDLs), each of which plays a different role in reverse cholesterol transport. In this study we used 2D-PAGGE to investigate the role of apoA-IV–containing lipoproteins in reverse cholesterol transport in native plasma. Incubation of 2D electrophoretograms with anti–apoA-IV antibodies identified up to three subclasses of particles. The smaller particle subclasses, LpA-IV-1 and LpA-IV-2, were found in every plasma sample. The largest particle subclass, LpA-IV-3, was observed in fewer than 10% of the plasmas analyzed. 2D-PAGGE of apoA-I–deficient plasma and apoA-I–depleted plasma and anti–apoA-I immunosubtracting 2D-PAGGE of normal plasma revealed that LpA-IV-1 and LpA-IV-2 do not contain apoA-I. The importance of LpA-IV-1 and LpA-IV-2 for uptake and esterification of cell-derived cholesterol was investigated using pulse-chase incubations of plasma with [
3
H]cholesterol-labeled fibroblasts followed by anti–apoA-I immunosubtracting 2D-PAGGE. During 1-minute pulse incubation with cells, [
3
H]cholesterol was taken up by γ-LpE >LpA-IV-1 >pre-β
1
-LpA-I >LpA-IV-2 (“>” denotes “more than”). During subsequent chase incubation without cells, proportionately less radioactivity disappeared from LpA-IV-1 and LpA-IV-2 than from pre-β
1
-LpA-I and γ-LpE. During 5-minute pulse incubations, radioactive cholesteryl esters were formed in pre-β
3
-LpA-I >α-LpA-I >LpA-IV-1 >LpA-IV-2. The fractional esterification rate was highest in pre-β
3
-LpA-I and lowest in α-LpA-I. Subsequent chase led to the disappearance of [
3
H]cholesteryl esters from pre-β
3
-LpA-I and, to a lesser extent, from LpA-IV-1 and LpA-IV-2 but to an increase of [
3
H]cholesteryl esters in α-LpA-I and LDL. Similar pulse-chase experiments with apoA-I–deficient plasma revealed that LpA-IV-1 and LpA-IV-2 take up and esterify cell-derived cholesterol even more effectively than in normal plasma. We conclude that LpA-IV-1 and LpA-IV-2 are apoA-I–free lipoproteins that are important contributors to reverse cholesterol transport.
Synergistic Effects of Fluid Shear Stress and Cyclic Circumferential Stretch on Vascular Endothelial Cell Morphology and Cytoskeleton
Authors: Zhao Shumin , Suciu Andreas , Ziegler Thierry , Moore James E. , Bürki Ernst , Meister Jean-Jacques , Brunner Hans R.
Published: June 2011

Abstract
The development of atherosclerosis is thought to be initiated by a dysfunctional state of the vascular endothelium. The proposal that mechanical forces play a role in the localization of this disease has led researchers to develop in vitro models to assess their effects on cultured endothelial cells. The arterial endothelium is exposed simultaneously to circumferential hoop stretch and wall shear stress, yet previous investigations have focused on the isolated effects of either cyclic stretch or shear stress. The influence of physiological levels of combined shear stress and hoop stretch on the morphology and F-actin organization of bovine aortic endothelial cells was investigated. Cells subjected for 24 hours to shear stresses higher than 2 dyne/cm
2
or to hoop stretch greater than 2% elongated significantly compared with unstressed controls and oriented along the direction of flow and perpendicular to the direction of stretch. Exposure to more than 4% stretch significantly enhanced the responses to shear stress. Both shear stress and hoop stretch induced formation of stress fibers that were aligned with the cells’ long axes. Simultaneous exposure to both stimuli appeared to enhance stress fiber size and alignment. These results indicate that shear stress and hoop stretch synergistically induce morphological changes in endothelial cells, which suggests that circumferential strain might modulate sensitivity of endothelial cells towards shear stress.
Effect of Diabetes on Lipoprotein Size
Authors: Singh Amareshwar T.K. , Rainwater David L. , Haffner Steven M. , VandeBerg John L. , Shelledy Wendy R. , Moore Perry H. , Dyer Thomas D.
Published: June 2011

Abstract
The effects of diabetes on lipoprotein particle sizes were assessed using samples from 94 subjects with non–insulin-dependent diabetes mellitus. From a larger population of nondiabetic subjects who showed normal glucose tolerance, we selected an exact match in terms of age, sex, and menopausal status. We designed a protocol to make nondenaturing gradient gels for the resolution of LDL subfractions and generated two measures of LDL size: diameter of the predominant LDL species and proportion of LDL cholesterol (LDL-C) in particles larger than 25.5 nm (large LDL-C). Similarly, we made two measures of HDL size, large HDL cholesterol (HDL-C) and large HDL–apoAI, which represent the proportion of HDL-C and apoAI, respectively, occurring on particles larger than HDL
3
. In pairwise comparisons, diabetes was associated with significantly (
P
<.004) smaller lipoprotein particles for all measures except large HDL-C. Each of the size measures was significantly and positively correlated with each of the others, suggesting that common metabolic mechanisms influence lipoprotein particle sizes across classes of lipoproteins. In addition, each of the size measures was correlated with a variety of measures of HDL and β-lipoprotein concentrations, which included HDL-C, LDL-C, triglycerides, and apoAI, apoB, and apoE. We used stepwise regression analyses to select from the measures of lipoprotein concentrations those independently correlated with each of the lipoprotein size measures. After adjusting for these metabolic correlates of lipoprotein size measures, we found the effect of diabetes on lipoprotein size measures was no longer significant except for a modest effect (
P
=.027) on large HDL–apoAI. These results suggest that diabetes alters aspects of lipoprotein metabolism that result in modification of lipoprotein particle sizes.
Triglycerides Are Major Determinants of Cholesterol Esterification/Transfer and HDL Remodeling in Human Plasma
Authors: Murakami Toru , Michelagnoli Silvia , Longhi Renato , Gianfranceschi Gemma , Pazzucconi Franco , Calabresi Laura , Sirtori Cesare R. , Franceschini Guido
Published: June 2011

Abstract
Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n=20) and isolated (HTG, n=20) or mixed hypertriglyceridemia (MHTG, n=20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37°C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG+MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (
r=
.722 and
r=
.642, respectively), CETR (
r=
.510 and
r=
.491, respectively), and HDL remodeling (
r=
.625 and
r=
.620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.
Osteopontin Is Not a Marker for Proliferating Human Vascular Smooth Muscle Cells
Authors: Newman Christopher M. , Bruun Birgitte C. , Porter Karen E. , Mistry Pramod K. , Shanahan Catherine M. , Weissberg Peter L.
Published: June 2011

AbstractOsteopontin (OP) is a secreted glycoprotein that contains the Arg-Gly-Asp (RGD) cell-binding sequence that binds calcium and is chemotactic and adhesive for rat vascular smooth muscle cells (VSMCs). OP gene expression is upregulated in cultured rat VSMCs in vitro and after balloon carotid injury in vivo, suggesting that OP may be a marker for proliferating VSMCs in vivo and in vitro. Our in situ hybridization studies of human atherosclerotic coronary vessels, however, have shown OP mRNA expression in plaque macrophages but not in VSMCs. The current study investigated OP mRNA expression in cultured human VSMCs and macrophages and in an organ culture model of neointima formation in human saphenous vein. OP mRNA expression was not detected by Northern blot analysis of total RNA from subconfluent or confluent cultures of human VSMCs of any passage maintained in normal growth medium or after stimulation with TGFβ1(20 ng/mL), angiotensin II (1 μmol/L), or basic fibroblast growth factor (10 ng/mL) but was just detectable after stimulation with activated vitamin D3(1 μmol/L). In contrast, cultured human macrophages expressed high levels of OP mRNA that were not dependent on lipid loading. OP mRNA was detected in isolated foci in all layers of saphenous veins maintained in organ culture for 14 days, including <2% of neointimal cells, a distribution that paralleled that of tissue macrophages. These results suggest that OP gene expression is not a marker for proliferation of human VSMCs in vitro and highlight a fundamental difference in the biology of human and rodent VSMCs.
Thrombin Induces the Redistribution and Acute Release of Tissue Factor Pathway Inhibitor From Specific Granules Within Human Endothelial Cells in Culture
Authors: Lupu Cristina , Lupu Florea , Dennehy Ulla , Kakkar Vijay V. , Scully Michael F.
Published: June 2011

Abstract
Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue factor (TF)–dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells show that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be of physiological importance in controlling TF-mediated thrombin generation.
Inhibition of Nitric Oxide Biosynthesis Promotes P-selectin Expression in Platelets
Authors: Murohara Toyoaki , Parkinson Scott J. , Waldman Scott A. , Lefer Allan M.
Published: June 2011

Abstract
Inhibition of NO synthesis promotes P-selectin expression on endothelial cells; however, the precise mechanism is unclear. Because NO has been shown to inhibit protein kinase C (PKC) activity, we examined the hypothesis that the NO synthase inhibitor
N
G
-nitro-
l
-arginine methyl ester (
l
-NAME) stimulates P-selectin expression on platelets via PKC activation. Ten-minute incubation with either phorbol 12-myristate 13-acetate (PMA), thrombin, or
l
-NAME significantly increased P-selectin expression on platelets (as assessed by flow-cytometric analysis) and PKC activity of platelet membranes. Increased P-selectin expression induced by either PMA, thrombin, or
l
-NAME was significantly attenuated by the selective PKC inhibitor UCN-01 (7-hydroxystaurosporine). Furthermore,
l
-NAME–induced P-selectin expression was significantly attenuated by either
l
-arginine, 8-bromo-cGMP, or sodium nitroprusside (SNP). Interestingly,
l
-NAME further potentiated P-selectin upregulation by thrombin.
l
-NAME, thrombin, and PMA also significantly increased polymorphonuclear leukocyte adherence to the coronary artery endothelium, an effect that was significantly attenuated by the anti–P-selectin monoclonal antibody PB1.3 or by UCN-01,
l
-arginine, 8-bromo-cGMP or SNP but not by
d
-arginine or the nonblocking anti–P-selectin monoclonal antibody NBP1.6. These results indicate that inhibition of NO synthesis induces rapid P-selectin expression, which appears to be at least partially mediated by PKC activation in platelets. Similar effects and mechanisms of
l
-NAME on P-selectin function were also observed in endothelial cells, another site of P-selectin expression. Thus, PKC activation may play an important role in cell-to-cell interaction when NO production is compromised.
Dominant Social Status and Contraceptive Hormone Treatment Inhibit Atherogenesis in Premenopausal Monkeys
Authors: Kaplan Jay R. , Adams Michael R. , Anthony Mary S. , Morgan Timothy M. , Manuck Stephen B. , Clarkson Thomas B.
Published: June 2011

Abstract
The stress of social subordination is associated with exacerbation of coronary artery atherosclerosis in premenopausal cynomolgus monkeys, possibly as a result of the ovarian dysfunction that reliably accompanies subordinate social status. The primary objective of the current study was to determine whether treatment with an oral contraceptive (OC) provides relative protection from development of atherosclerotic plaques, especially among animals made vulnerable to atherosclerosis by social subordination. In the present study, 193 adult female monkeys
(Macaca fascicularis)
were placed in social groups of 5 or 6 animals each. Half of the animals were then fed an atherogenic diet to which had been added a triphasic OC, while the remainder received only the atherogenic diet. At the end of 26 months, atherosclerosis was measured in an iliac artery biopsy taken from each monkey. The results demonstrated that among untreated animals subordinate individuals developed significantly more atherosclerosis than did their dominant counterparts (
P
<.01); however, OC treatment inhibited atherosclerosis in subordinate animals (
P
<.05) and eliminated the difference between dominant and subordinate animals that was observed in the untreated condition. Subordinate social status and OC treatment were both associated with reduced plasma concentrations of HDL cholesterol (
P
<.01 for both), and subordinate monkeys also had elevations in LDL cholesterol plus VLDL cholesterol (
P
<.01). Nonetheless, the interaction between social status and OC treatment remained significant even after covariance adjustment for variation in plasma lipids. Taken together, these results suggest that social subordination worsens, whereas OC treatment inhibits, atherosclerosis, and that these effects are independent of concomitant variability in plasma lipids.
Influence of Diets Rich in Saturated and Omega-6 Polyunsaturated Fatty Acids on the Postprandial Responses of Apolipoproteins B-48, B-100, E, and Lipids in Triglyceride-Rich Lipoproteins
Authors: Bergeron Nathalie , Havel Richard J.
Published: June 2011

Abstract
The effects of diets rich in saturated fatty acids (SFA) (total polyunsaturated fatty acids [PUFA] [g]/total SFA [g][P/S ratio], 0.2) or omega-6 PUFA (P/S ratio, 1.3) on the postprandial response of triglyceride-rich lipoproteins (TRL) was determined in normolipidemic young men. After 15 and 29 days of diet intervention, the postabsorptive concentrations of apolipoprotein (apo) B-48 and apoB-100 were higher in the SFA group than in the PUFA group, but the absolute increase in apoB-48 was similar 3 hours after a challenge meal containing one third of daily energy and returned to postabsorptive values at 6 hours; this response was closely coupled to that of TRL triglycerides. In both groups, the percent increase in TRL apoB-48 and triglycerides was greater after the PUFA meal than after the SFA meal. The concentration of TRL apoB-100 also increased at 3 hours in both diet groups but returned to postabsorptive values at 6 hours only in those fed the PUFA diet; in the SFA group, apoB-100 remained high at 6 hours and fell below postabsorptive values only 9 hours after the meal. This apoB-100 response was affected primarily by the fatty acid composition of the diet and not by that of the challenge meal. The postprandial response of apoB-100 was closely coupled to that of cholesterol and apoE. These observations suggest that in healthy young men, neither the fatty acid composition of the diet nor that of the challenge meal affects the clearance of chylomicron remnants after a fat-containing meal. By contrast, the postprandial accumulation of hepatogenous TRL is prolonged in individuals fed a diet rich in SFA.
Association of Postprandial Triglyceride and Retinyl Palmitate Responses With Asymptomatic Carotid Artery Atherosclerosis in Middle-aged Men and Women
Authors: Sharrett A. Richey , Chambless Lloyd E. , Heiss Gerardo , Paton Catherine C. , Patsch Wolfgang
Published: June 2011

Abstract
Blood lipid alterations after a fatty meal may be atherogenic, but there is little information regarding their associations with disease independent of fasting lipids. Asymptomatic atherosclerosis cases (n=229) and 373 control subjects free of atherosclerosis, as defined by carotid intima-media thickness on ultrasound images, were given a fatty meal with vitamin A, followed by 3.5- and 8-hour measurements of triglycerides (TGs), TG-rich lipoprotein TGs, apoproteinB48, and retinyl palmitate. Among white men and women but not among blacks, case status was associated with greater postprandial responses of TGs and TG-rich lipoprotein TGs, but only in nonobese persons (body mass index <30 kg/m
2
). The associations were strong and significant after controlling for coronary risk factors (odds ratio, ≈2.0) and fasting TGs (odds ratio, 1.5). Associations with other postprandial lipid measurements did not persist after controlling for fasting lipids. Elevated postprandial TGs appear to be an independent risk factor for carotid intimal thickening in nonobese whites. The lack of such a relation in obese subjects and the lipid profile they manifest suggest that postprandial TGs must be accompanied by accumulation of TG-rich lipoprotein remnants to be atherogenic.
Increased Elastin-Degrading Activity and Neointimal Formation in Porcine Aortic Organ Culture
Authors: Oho Shinichi , Daley Sandra J. , Koo Edward W.Y. , Childs Tim , Gotlieb Avrum I. , Rabinovitch Marlene
Published: June 2011

AbstractWe investigated the association between tissue elastolytic activity and the development of neointimal formation using a previously described porcine aortic organ culture. Neointimal formation is associated with the presence of intact endothelium (nondenuded cultures) but is markedly reduced if endothelial cells are removed (denuded cultures). In nondenuded organ cultures, elastolytic activity assessed by using [3H]elastin increased sixfold at day 3 after initiation of the culture (P<.01), a time earlier than the previously published increase in intimal smooth muscle cells (ISMCs). Elastolytic activity did not increase from day 3 to day 7 despite doubling of ISMCs but did double by day 14 (P<.01) and remained elevated to day 28, correlating with increases in ISMCs. In denuded organ cultures, elastolytic activity was much lower than in nondenuded organ cultures at day 3 (P<.05) but increased fivefold in the presence of nondenuded organ culture conditioned medium (P<.01). Addition of α1-proteinase inhibitor for 14 days caused a 60% decrease in elastolytic activity in nondenuded organ cultures and a 27% reduction in ISMCs compared with untreated controls (P<.05 for both). The elastolytic activity, resolved as lytic bands on an elastin substrate gel, reflected candidate enzymes, one at 76 kD and perhaps a doublet at 43 and 50 kD. Our study suggests that endothelial cells release a soluble agent that enhances elastin-degrading activity in the aorta and may at least partially account for the initiation of neointimal formation.
Aldose Reductase Inhibitor Prevents Hyperproliferation and Hypertrophy of Cultured Rat Vascular Smooth Muscle Cells Induced by High Glucose
Authors: Yasunari Kenichi , Kohno Masakazu , Kano Hiroaki , Yokokawa Koji , Horio Takeshi , Yoshikawa Junichi
Published: June 2011

Abstract
Vascular remodeling is a key process in the pathophysiology of atherosclerosis. Recent evidence suggests that high glucose levels may function as a vascular smooth muscle growth and proliferation–promoting substance. To explore the role of the polyol pathway in this process, we examined the effect of an aldose reductase inhibitor (ARI), epalrestat, on the growth characteristics of cultured rat vascular smooth muscle cells (VSMCs). Epalrestat (10 nmol/L, 1 μmol/L) significantly suppressed the high glucose–induced proliferative effect as measured by [
3
H]thymidine incorporation by 67% and 82% in cell number, suggesting ARI as an antimitogenic factor. In VSMCs, epalrestat (10 nmol/L, 1 μmol/L) significantly suppressed the high glucose–induced incorporation of [
3
H]leucine by 45% and 58% with the concomitant reduction of the cell size estimated by flowcytometry. Epalrestat (1 μmol/L) also suppressed high glucose–induced intracellular NADH/NAD
+
increase and membrane-bound protein kinase C activation. These results indicate that this ARI possesses an antiproliferative and antihypertrophic action on VSMCs induced by high glucose possibly through protein kinase C suppression.
Genetic Determination of Cartilaginous Metaplasia in Mouse Aorta
Authors: Qiao Jian-Hua , Fishbein Michael C. , Demer Linda L. , Lusis Aldons J.
Published: June 2011

Abstract
Calcification frequently occurs in atherosclerotic plaques in humans, but the cellular and genetic factors contributing to this pathological trait are unknown. We previously reported that the arterial calcification among inbred strains is genetically determined, and we now report that cartilaginous metaplasia, associated with the presence of arterial chondrocytes that express type II collagen, may underlie this calcification. Both uncalcified and calcified cartilaginous metaplasia were often colocalized with aortic atheromatous lesions and calcification, and clear genetic differences were observed in the occurrence of aortic cartilaginous metaplasia among inbred strains. Analysis of a genetic cross between strains C57BL/6J (exhibiting aortic cartilaginous metaplasia) and C3H/HeJ (no aortic cartilaginous metaplasia) revealed a recessive inheritance pattern; thus, F
1
mice were entirely devoid of cartilaginous metaplasia, in common with the C3H/HeJ parental strain. Analyses of an F
2
cross and a set of recombinant inbred strains derived from parental strains C57BL/6J and C3H/HeJ were consistent with a major gene effect exhibiting incomplete penetrance. The occurrence of aortic calcification was correlated with the occurrence of cartilaginous metaplasia in these genetic crosses, suggesting a link between the traits. Finally, we observed widespread calcified cartilaginous metaplasia within spontaneous atherosclerotic lesions in mice targeted for a null mutation in the apoE gene, suggesting that cartilaginous metaplasia is a potential pathway for artery wall calcification associated with the atherosclerotic plaque.
Activation of Protein Kinase C Increases Adenosine Production in the Hypoxic Canine Coronary Artery Through the Extracellular Pathway
Authors: Minamino Tetsuo , Kitakaze Masafumi , Komamura Kazuo , Node Koichi , Takeda Hiroshi , Inoue Michitoshi , Hori Masatsugu , Kamada Takenobu
Published: June 2011

AbstractBoth ischemia and hypoxia increase adenosine production in the heart. This study tested whether hypoxia increases adenosine production in the coronary artery via ecto-5′-nucleotidase and the role of protein kinase C in this condition. Canine left circumflex coronary artery was rapidly removed and incubated in 10 mL Krebs-Henseleit solution for 30 minutes. The Krebs-Henseleit solution contained 5′-iodotubercidin and 2′-deoxycoformycin, which inhibit adenosine kinase and adenosine deaminase, respectively. Adenosine production was measured in intact coronary arteries under normoxic conditions (16.2±1.2 pmol/mg protein). Adenosine production was reduced by 27% after removal of endothelium. Ecto-5′-nucleotidase activity of coronary arteries with and without endothelium was 51±6 and 41±4 nmol/mg protein per minute under normoxic conditions. Hypoxia increased adenosine production to 27.0±2.3 and 20.0±0.8 pmol/mg protein with and without endothelium. Hypoxia also increased ecto-5′-nucleotidase activity of coronary arteries with and without endothelium (74±8 and 53±5 nmol/mg protein per minute;P<.05). Increases in adenosine production under hypoxic conditions were blunted by both an inhibitor of ecto-5′-nucleotidase and inhibitors of protein kinase C. Activation of ecto-5′-nucleotidase was blunted by an inhibitor of protein kinase C. These results indicate that hypoxia increased extracellular adenosine production and activated ecto-5′-nucleotidase via activation of protein kinase C in coronary arterial smooth muscle and endothelial cells. Increased adenosine production in coronary arteries during hypoxia may contribute to coronary vasodilation and cardioprotection against ischemic injury.
The Effect of α-Tocopherol Supplementation on LDL Oxidation
Authors: Jialal Ishwarlal , Fuller Cindy J. , Huet Beverley A.
Published: June 2011

Abstract
Because much data have accrued to support the concept that oxidatively modified LDL (Ox-LDL) can promote atherogenesis, the role of antioxidants in decreasing LDL oxidation has assumed great importance. High-dose α-tocopherol supplementation in humans decreases the susceptibility of LDL to oxidation. Hence, the aim of the present study was to ascertain the minimum dose of α-tocopherol that would decrease the susceptibility of LDL to oxidation. The effect of α-tocopherol in doses of 60, 200, 400, 800, and 1200 IU/d on copper-catalyzed LDL oxidation was tested in a randomized placebo-controlled study over 8 weeks. There were eight subjects in each group. Oxidation of LDL was monitored by measuring the formation of conjugated dienes and lipid peroxides by the thiobarbituric acid–reacting substances (TBARS) assay over an 8-hour time course at baseline and after 8 weeks of supplementation. Neither placebo nor any of the doses of α-tocopherol resulted in any side effects or exerted an adverse effect on the plasma lipoprotein profile. However, there was a dose-dependent increase in plasma and lipid-standardized α-tocopherol levels with increasing doses of α-tocopherol supplementation. LDL α-tocopherol appeared to follow a similar trend. When the time-course curves of LDL oxidation and the kinetics of LDL oxidation were examined, there was no significant effect at 8 weeks compared with baseline in the groups that received placebo or α-tocopherol 60 or 200 IU/d. However, in the groups that received at least 400 IU/d α-tocopherol, there was a decreased susceptibility of LDL to oxidation, as shown by the mean levels in the time-course curves, prolongation in the lag phase, and a decrease in the oxidation rate. Furthermore, both plasma and LDL α-tocopherol correlated significantly with the lag phase of oxidation and inversely with the oxidation rate. The results of the present study show that the minimum dose of α-tocopherol needed to significantly decrease the susceptibility of LDL to oxidation is 400 IU/d.