bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms Regulating LDL Metabolism in Subjects on Peroral and Transdermal Estrogen Replacement Therapy
Authors: Karjalainen Anna , Heikkinen Jorma , Savolainen Markku J. , Bäckström Ann-Christine , Kesäniemi Y. Antero
Published: June 2011

Abstract
—To study the mechanisms of low density lipoprotein (LDL) cholesterol lowering by peroral and transdermal estrogen replacement therapy (ERT), 79 hysterectomized postmenopausal women aged 48 to 62 years were randomized in a double-blind double-dummy trial to receive either peroral estradiol valerate (2 mg/d) or transdermal estradiol gel (1 mg/d) for 6 months. Plasma LDL cholesterol decreased from 4.19±0.83 (mean±SD) to 3.39±0.78 mmol/L (
P
<0.001) in the peroral group and from 4.11±0.86 to 3.72±0.78 mmol/L (
P
<0.001) in the transdermal estrogen group. Peroral estrogen did, but transdermal treatment did not, enhance the fractional catabolic rate (FCR) and production of LDL apolipoprotein B (apoB). However, the decrease of LDL cholesterol was related to an increase in FCR for LDL apoB on both peroral and transdermal ERT (
r
=−0.645,
P
<0.001 and
r
=−0.627,
P
<0.001, respectively). These changes were associated with changes in the serum estrogen level. Both therapies reduced absorption of dietary cholesterol by 6% to 10% (
P
<0.05). The effects of estrogen were not modified by the polymorphisms of apoE and apoB or cholesterol 7α-hydroxylase. In conclusion, the ERT-induced LDL cholesterol–lowering effect is related to changes in estrogen level, which presumably enhance LDL receptor activity, which is manifested as an increase in FCR for LDL apoB. The small decrease in the absorption efficiency of dietary cholesterol does not seem to contribute largely to the cholesterol lowering on either transdermal or peroral ERT.
Major Reduction in Plasma Lp(a) Levels During Sepsis and Burns
Authors: Mooser Vincent , Berger Mette M. , Tappy Luc , Cayeux Christine , Marcovina Santica M. , Darioli Roger , Nicod Pascal , Chioléro René
Published: June 2011

Abstract
—Plasma levels of lipoprotein(a) [Lp(a)], an atherogenic particle, vary widely between individuals and are highly genetically determined. Whether Lp(a) is a positive acute-phase reactant is debated. The present study was designed to evaluate the impact of major inflammatory responses on plasma Lp(a) levels. Plasma levels of C-reactive protein (CRP), low density lipoprotein cholesterol, Lp(a), and apolipoprotein(a) [apo(a)] fragments, as well as urinary apo(a), were measured serially in 9 patients admitted to the intensive care unit for sepsis and 4 patients with extensive burns. Sepsis and burns elicited a major increase in plasma CRP levels. In both conditions, plasma concentrations of Lp(a) declined abruptly and transiently in parallel with plasma low density lipoprotein cholesterol levels and closely mirrored plasma CRP levels. In 5 survivors, the nadir of plasma Lp(a) levels was 5- to 15-fold lower than levels 16 to 18 months after the study period. No change in plasma levels of apo(a) fragments or urinary apo(a) was noticed during the study period. Turnover studies in mice indicated that clearance of Lp(a) was retarded in lipopolysaccharide-treated animals. Taken together, these data demonstrate that Lp(a) behaves as a negative acute-phase reactant during major inflammatory response. Nongenetic factors have a major, acute, and unexpected impact on Lp(a) metabolism in burns and sepsis. Identification of these factors may provide new tools to lower elevated plasma Lp(a) levels.
Fibroblast Growth Factor Plays a Critical Role in SM22α Expression During
Xenopus
Embryogenesis
Authors: Oka Toru , Shiojima Ichiro , Monzen Koshiro , Kudoh Sumiyo , Hiroi Yukio , Shiokawa Koichiro , Asashima Makoto , Nagai Ryozo , Yazaki Yoshio , Komuro Issei
Published: June 2011

Abstract
—Although smooth muscle cells (SMCs) are critical components of the circulatory system, the regulatory mechanisms of SMC differentiation remain largely unknown. In the present study, we examined the mechanism of SMC differentiation by using
Xenopus laevis
SM22α (XSM22α) as an SMC-specific marker. XSM22α cDNA contained a 600-bp open reading frame, and the predicted amino acid sequences were highly conserved in evolution. XSM22α transcripts were first detected in heart anlage, head mesenchyme, and the dorsal side of the lateral plate mesoderm at the tail-bud stage, possibly representing the precursors of muscle lineage. At the tadpole stage, XSM22α transcripts were restricted to the vascular and visceral SMCs. XSM22α was strongly induced by basic fibroblast growth factor (FGF) in animal caps. Although expressions of
Xenopus
cardiac actin were not affected by the expression of a dominant-negative FGF receptor, its injection dramatically suppressed the XSM22α expression. These results suggest that XSM22α is a useful molecular marker for the SMC lineage in
Xenopus
and that FGF signaling plays an important role in the induction of XSM22α and in the differentiation of SMCs.
Upregulation and Activation of Stat6 Precede Vascular Smooth Muscle Cell Proliferation in Carotid Artery Injury Model
Authors: Baetta Roberta , Soma Maurizio , De-Fraja Claudio , Comparato Carmen , Teruzzi Chiara , Magrassi Lorenzo , Cattaneo Elena
Published: June 2011

Abstract
—The role of signal transducers and activators of transcription (STAT) proteins in modulating proliferation and differentiation of various cell types in the hematopoietic system and the central nervous system has been well established. In contrast, the pathophysiological role of these proteins in vascular proliferative diseases has remained unproven, despite in vitro observations emphasizing the involvement of the STAT system in mediating vascular smooth muscle cell (VSMC) proliferation. On the basis of our previous observations demonstrating the occurrence of a specific modulation of Stat6 protein during the proliferative, migratory, and differentiation phases of the developing brain, we investigated whether Stat6 protein is present and modulated in arterial tissue challenged by perivascular injury. The time course of expression and localization of Stat6 after arterial injury was analyzed by immunohistochemistry, Western blot analysis, and confocal microscopy. Six hours after injury, the expression of Stat6 was markedly increased. This overexpression preceded the onset of VSMC proliferation and was downregulated starting from 7 days after injury, coincident with the decline of VSMC proliferation. Moreover, early after injury, Stat6 was predominantly localized at the nuclear level, denoting its functional activation. Conversely, Stat6 staining at later time points was largely cytosolic, suggesting silencing effects of this signaling pathway. These data indicate that Stat6 signaling may contribute to the modifications of gene expression underlying VSMC activation in the context of acute vascular proliferative diseases.
Fibronectin in an Extracellular Matrix of Cultured Endothelial Cells Supports Platelet Adhesion via Its Ninth Type III Repeat
Authors: Beumer Sara , Heijnen-Snyder Glenda J. , IJsseldijk Martin J. W. , de Groot Philip G. , Sixma Jan J.
Published: June 2011

Abstract
—We investigated the involvement of different domains of fibronectin in mediating platelet adhesion to fibronectin in the extracellular matrix (ECM) of cultured endothelial cells under flow conditions. Polyclonal anti-fibronectin antibodies were absorbed with Sepharose to which no protein, intact fibronectin, or different fibronectin fragments had been coupled to obtain supernatants (Sups) (Sup
0
, Sup
FN
, and Sup
name of the fragment
, respectively) from which a specific part of the antibodies had been removed. Treatment of the ECM before perfusion with Sup
0
resulted in a 36% decrease in platelet coverage, whereas treatment with Sup
FN
resulted in maximal adhesion. Treatment of the ECM with supernatants from which antibodies directed against the gelatin- or heparin-binding domain had been removed showed the same inhibition as treatment with Sup
0
. Removal of antibodies directed to the 120-kDa cell-binding domain resulted in a level of adhesion equal to the level found when the ECM was treated with Sup
FN
. Further analysis of this central region showed that only treatment with supernatants from which antibodies directed to the ninth type III repeat (III-9) of fibronectin had been removed resulted in a significantly higher adhesion than treatment with Sup
0
. Studies of adhesion to the fragments themselves showed that only fragments containing III-10 were able to support adhesion. Mutation of the Arg-Gly-Asp (RGD) sequence into Arg-Gly-Glu (RGE) in one of those fragments resulted in a complete loss of adhesive capacity. These data suggest that platelet adhesion to fibronectin in the ECM depends on III-9, whereas III-10 does not seem to be required. For platelet adhesion to isolated fibronectin, an intact RGD sequence seems to be crucial.
1999 George Lyman Duff Memorial Lecture
Authors: Tall Alan R. , Jiang Xian-cheng , Luo Yi , Silver David
Published: June 2011

Abstract
—Plasma high density lipoprotein (HDL) levels show an inverse relationship to atherogenesis, in part reflecting the role of HDL in mediating reverse cholesterol transport. The transfer of HDL cholesterol to the liver involves 3 catabolic pathways: the indirect, cholesteryl ester transfer protein (CETP)–mediated pathway, the selective uptake (scavenger receptor BI) pathway, and a particulate HDL uptake pathway. The functions of the lipid transfer proteins (CETP and phospholipid transfer protein) in HDL metabolism have been elucidated by genetic approaches in humans and mice. Human CETP deficiency is associated with increased HDL levels but appears to increase coronary artery disease risk. Phospholipid transfer protein deficiency, produced by gene knockout in mice, results in decreased HDL levels, reflecting decreased transfer of phospholipids from triglyceride-rich lipoproteins into HDL. Obese (
ob
/
ob
) mice have markedly increased HDL levels and represent an interesting model of defective HDL catabolism in the liver. In hepatocytes of wild-type mice, there is extensive uptake and resecretion of HDL and selective uptake of cholesteryl ester from HDL during recycling. In
ob
/
ob
mice, these processes are defective, suggesting that HDL recycling plays an important role in holo-HDL catabolism, selective uptake, and the determination of plasma HDL levels.
Localization of CD9, an Enhancer Protein for Proheparin-Binding Epidermal Growth Factor–Like Growth Factor, in Human Atherosclerotic Plaques
Authors: Nishida Makoto , Miyagawa Jun-ichiro , Yamashita Shizuya , Higashiyama Shigeki , Nakata Atsuyuki , Ouchi Noriyuki , Tamura Ritsu , Yamamori Katsumi , Kihara Shinji , Taniguchi Naoyuki , Matsuzawa Yuji
Published: June 2011

Abstract
—Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF family, has a potent mitogenic activity for vascular smooth muscle cells (SMCs). We previously reported that HB-EGF is involved in atherogenesis of human aorta and coronary arteries. ProHB-EGF (the membrane-anchored form of HB-EGF) has also been demonstrated to possess a mitogenic activity, which is ≈30-fold increased when coexpressed with CD9 in mouse L cells. Thus, in the process of atherogenesis, CD9 may be involved in the proliferation of SMCs. We immunohistochemically investigated the localization of CD9 and proHB-EGF in the human aorta and coronary arteries. In normal aorta and coronary arteries, CD9 immunostaining was virtually negative, whereas proHB-EGF immunostaining was positive, especially in the arteries of babies. In contrast, in atherosclerotic lesions, some intimal SMCs were strongly positive for CD9 and proHB-EGF immunostaining. The juxtacrine growth activities of human aortic SMCs were inhibited in vitro by adding neutralization antibodies for CD9 or adding the specific inhibitor of HB-EGF. Besides, coexpressed CD9 and proHB-EGF cells markedly incorporated [
3
H]thymidine into the SMCs. CD9 is localized immunohistochemically in the SMCs of the atherosclerotic aorta and coronary arteries. CD9, when coexpressed with proHB-EGF, enhances proHB-EGF activities for SMC growth in a so-called juxtacrine manner in vitro and may be involved in atherogenesis.
Endothelin-1 and Smooth Muscle Cells
Authors: Fei Jianwei , Viedt Christiane , Soto Ubaldo , Elsing Christoph , Jahn Lothar , Kreuzer Jörg
Published: June 2011

Abstract
—Endothelin-1 (ET-1) has been proposed to contribute to atherogenesis and plaque rupture in coronary heart disease through activation of mitogen-activated protein kinases (MAPKs) in smooth muscle cells (SMCs). Reactive oxygen species (ROS) have been shown to be important signal transduction molecules in SMCs. Thus, the present study aimed to assess the role of ROS in ET-1–mediated activation of c-Jun amino-terminal kinase (JNK) and extracellular signal–regulated kinase (ERK) 1/2. Rat SMCs were exposed to ET-1 over time at concentrations from 10
−6
to 10
−10
mol/L, and MAPK activity was quantified. Activation of JNK and ERK was observed with a maximum stimulation at 10
−7
mol/L ET-1. JNK and ERK were activated by ET-1 binding to a single receptor (ET-1A) but differed in their downstream mechanisms: only JNK activation was sensitive to the radical scavenger
N
-acetylcysteine and diphenylene iodonium, an inhibitor of NADPH oxidase, indicating a role for ROS. The downstream MAPK effector and proinflammatory transcription factor, the activator protein-1 complex, was maximally activated 2 hours after the addition of ET-1. It was mainly composed of the JNK substrate c-Jun, and activation was also dependent on ROS formation. We suggest that plaque activation by ET-1 can be mediated through ROS. It can be hypothesized that the clinical benefit of antioxidants in the treatment of atherogenesis may partially depend on neutralization of ET-1–mediated ROS production.
Induction of IG9 Monocyte Adhesion Molecule Expression in Smooth Muscle and Endothelial Cells After Balloon Arterial Injury in Cholesterol-Fed Rabbits
Authors: Calderon Tina M. , Gertz S. David , Sarembock Ian J. , Berliner Judith A. , Fallon John T. , Taubman Mark B. , Berman Joan W.
Published: June 2011

Abstract
—The expression of monocyte-specific adhesion molecules and chemokines by cell types within the vessel wall plays an important role in foam cell accumulation during atherosclerotic plaque development. We previously identified IG9, a novel monocyte adhesion protein that is expressed on endothelial cells (ECs) overlying human and rabbit advanced atherosclerotic plaques. The present study was designed to determine the temporal and spatial expression of IG9 and the chemokine, monocyte chemoattractant protein-1 (MCP-1), after balloon injury with (double injury) or without (single injury) prior air desiccation EC injury in the femoral arteries of rabbits fed a high-cholesterol diet. By immunohistochemical analyses, intense reactivity with monoclonal antibodies to IG9 and MCP-1 was detected 24 hours after single injury in medial smooth muscle cells (SMCs) and in SMCs of adventitial microvessels. However, monocyte infiltration of the tunica media was minimal or not detected in these sections. IG9 and MCP-1 antibody reactivity in vessel sections 28 days after single injury and 24 hours, 7 days, and 28 days after double injury was localized to medial and neointimal SMCs, foam cells, and luminal ECs overlying the plaques. Uninjured rabbit (cholesterol or normal diet) vessel sections exhibited minimal IG9 and MCP-1 immunostaining. In vitro studies using human aortic SMCs demonstrated IG9 protein induction after 24 hours of treatment with platelet-derived growth factor-BB and interferon-γ or epidermal growth factor. IG9 expression was further increased by pretreatment of SMCs with the proatherogenic lipid, minimally oxidized low density lipoprotein. After balloon injury (24 hours), IG9 is induced in vascular SMCs before the detectable accumulation of monocytes within the vessel wall. Thus, the expression of IG9 by SMCs as well as by ECs may be an important factor in the accumulation of foam cells in atherosclerotic plaque development after arterial injury.
Antithrombotic Effect of Platelet Glycoprotein Ib–Blocking Monoclonal Antibody Fab Fragments in Nonhuman Primates
Authors: Cauwenberghs Nancy , Meiring Muriel , Vauterin Stephan , van Wyk Veronika , Lamprecht Seb , Roodt Jan P. , Novák Levente , Harsfalvi Jolan , Deckmyn Hans , Kotzé Harry F.
Published: June 2011

Abstract
—Platelet adhesion in arterial blood flow is mainly supported by the platelet receptor glycoprotein (GP) Ib, which interacts with von Willebrand factor (vWF) that is bound to collagen at the site of vessel wall injury. Antibody 6B4 is a monoclonal antibody (MoAb) raised against purified human GPIb. MoAb 6B4 inhibits both ristocetin- and botrocetin-induced, vWF-dependent human platelet agglutination. MoAb 6B4 furthermore blocks shear-induced adhesion of human platelets to collagen I. We studied the antithrombotic effect of this inhibitory murine MoAb 6B4 in a baboon model of arterial thrombosis. When injected into baboons, intact IgG and its F(ab′)
2
fragments caused almost immediate thrombocytopenia, whereas injection of the Fab fragments alone did not. Fab fragments were subsequently used to investigate their in vivo effect on platelet deposition onto a thrombogenic device, consisting of collagen-rich, glutaraldehyde-fixed bovine pericardium (0.6 cm
2
), at a wall shear rate ranging from 700 to 1000 s
−1
. Baboons were either pretreated with Fabs to study the effect of inhibition on platelet adhesion or treated 6 minutes after placement of the thrombogenic device to investigate the effect on interplatelet cohesion. Pretreatment of the animals with bolus doses ranging from 80 to 640 μg/kg Fab fragments significantly reduced
111
In-labeled platelet deposition onto the collagen surface by ≈43% to 65%. Only the highest dose caused a significant prolongation (doubling) of the bleeding time. Ex vivo ristocetin-induced platelet agglutination was equally reduced. Treatment with a bolus of 110 μg/kg Fab fragments after a thrombus was allowed to form for 6 minutes had no effect on further platelet deposition. We therefore conclude that Fab fragments or derivatives of inhibitory anti-GPIb antibodies may be useful compounds to prevent thrombosis.
Postmenopausal Hormone Replacement Therapy Increases Coagulation Activity and Fibrinolysis
Authors: Teede Helena J. , McGrath Barry P. , Smolich Joseph J. , Malan Erica , Kotsopoulos Dimitra , Liang Yu-Lu , Peverill Roger E.
Published: June 2011

Abstract
—Hormone replacement therapy (HRT) appears to be cardioprotective in postmenopausal women; however, concerns exist over its thrombogenic effects. To address the effects of combined HRT on coagulation and fibrinolysis, we have measured circulating markers of these processes in a double-blind placebo-controlled trial. Forty-two healthy postmenopausal women aged 50 to 75 years received continuous combined HRT with 2 mg estradiol+1 mg norethisterone or placebo for 6 weeks. Hormone profiles were measured at baseline, and lipid and hemostatic parameters were measured at baseline and after 6 weeks of therapy. Baseline characteristics were similar in the 2 groups. With change from baseline the main outcome measure
,
HRT increased the markers of coagulation (prothrombin fragments 1+2, 0.20±0.06 versus 0.06±0.04 nmol/L,
P
=0.0005; soluble fibrin, 2.3±0.4 versus 0.25±0.3 μg/mL,
P
=0.0004), reduced plasma fibrinolytic inhibitory activity (plasminogen activator inhibitor-1, −0.67±0.16 versus 0.24±0.21 U/mL,
P
=0.002), and increased fibrinolysis (D-dimer, 24±12 versus −6±8 ng/mL,
P
=0.04) compared with placebo. Increases in soluble fibrin and D-dimer were positively correlated (
r
=0.59,
P
=0.02), but changes in plasminogen activator inhibitor-1 and D-dimer were unrelated. Although baseline hemostatic and lipid parameters were correlated, there were no associations between changes in hemostatic markers and lipids after treatment. Short-term oral combined continuous HRT (estradiol and norethisterone) increased thrombin and fibrin generation, reduced plasma fibrinolytic inhibitory activity, and increased fibrinolysis. Enhanced fibrinolysis was related to increased fibrin generation but not reduced plasma fibrinolytic inhibitory activity. Coagulation activation may partly explain the increases in venous thrombosis and cardiovascular events reported with the use of combined HRT.
Enhanced H
2
O
2
-Induced Cytotoxicity in “Epithelioid” Smooth Muscle Cells
Authors: Li Wei-Gen , Miller Francis J. , Brown Michael R. , Chatterjee Papri , Aylsworth Gregory R. , Shao Jianqiang , Spector Arthur A. , Oberley Larry W. , Weintraub Neal L.
Published: June 2011

Abstract
—Vascular smooth muscle cells (SMCs) are phenotypically diverse. Although most medial SMCs can be classified as “fusiform,” others are of the “epithelioid” phenotype. Proliferation and apoptosis of epithelioid SMCs may contribute importantly to neointimal formation and regression, respectively. Because reactive oxygen species (ROS) are increased in vascular injury and can induce apoptosis of SMCs, we compared the effects of ROS on epithelioid and fusiform SMCs. Epithelioid and fusiform SMC lines were clonally isolated from rat aortic media and studied under similar conditions and passage numbers. H
2
O
2
produced dose- and time-dependent cytotoxicity that was enhanced in epithelioid compared with fusiform cells. After 24-hour exposure to 50 μmol/L H
2
O
2
, epithelioid cell numbers were reduced by 34±5% versus a 3±5% (
P
<0.05) reduction in fusiform cell numbers. Similar results were obtained whether H
2
O
2
was administered to growth-arrested or growing cells or when epithelioid and fusiform cells were exposed to extracellular O
2
.−
. To investigate whether apoptosis contributed to enhanced ROS-induced cytotoxicity in epithelioid SMCs, terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL) staining was performed. The incidence of TUNEL positivity was 5-fold increased in epithelioid versus fusiform SMCs after treatment with 50 μmol/L H
2
O
2
(19±1% epithelioid versus 5±1% fusiform,
P
<0.05). Enhanced H
2
O
2
-induced apoptosis in epithelioid SMCs was confirmed by DNA laddering. Furthermore, when balloon-injured aortas were exposed to H
2
O
2
ex vivo, enhanced apoptosis was observed in neointimal compared with medial SMCs. These results suggest that epithelioid SMCs exhibit enhanced sensitivity to ROS-induced apoptosis, which may play an important role in neointimal regression.
Increased Hyaluronan and Hyaluronidase Production and Hyaluronan Degradation in Injured Aorta of Insulin-Resistant Rats
Authors: Chajara Abdesslam , Raoudi Maha , Delpech Bertrand , Leroy Marcelle , Basuyau Jean Pierre , Levesque Hervé
Published: June 2011

Abstract
—Diabetic patients have a greater incidence of restenosis, which has been shown to be related to exaggerated intimal hyperplasia. Hyaluronan (HA) has been shown to be closely involved in arterial smooth muscle cell proliferation and migration, which provoke intimal hyperplasia after balloon catheter injury. Our aim was to determine the effect of fructose feeding, which produces certain characteristics of non–insulin-dependent diabetes (ie, insulin resistance, hyperinsulinemia, and hypertriglyceridemia), on production of HA and hyaluronidase and degradation of HA in rat aorta. Treated rats received fructose (25% in tap water) 12 weeks before balloon catheter injury and 14 days afterward. Fructose-fed rats had hyperinsulinemia and hypertriglyceridemia. Injury increased intima-media wet weight (7.5%) and DNA content (20%) in control rats. This increase was significantly greater in fructose-fed rats (22% for wet weight and 34% for DNA content) and was associated with greater HA and hyaluronidase production (123% and 41%, respectively) than in control rats (49% and 7%, respectively). Determination of HA molecular mass showed that balloon catheter injury increased the number of HA fragments in the aorta of control rats. Normal aorta of fructose-fed rats contained more HA fragments than that of control rats. Injury to the aorta of fructose-fed rats increased HA fragments and induced the appearance of a very-high-molecular-mass (>2000 kDa) HA. In conclusion, fructose treatment, which induced hyperinsulinemia and hypertriglyceridemia, increased HA and hyaluronidase production and HA degradation in injured aorta. This finding suggests that HA, which has been shown to play a crucial role in proliferation and migration of arterial smooth muscle cells, may be involved in the promotional effect of long-term fructose feeding on arterial wall reaction to injury.
Nitric Oxide Enhances Expression and Shedding of Tumor Necrosis Factor Receptor I (p55) in Endothelial Cells
Authors: Okuyama Masaki , Yamaguchi Seiji , Yamaoka Minako , Nitobe Joji , Fujii Satoshi , Yoshimura Tetsuhiko , Tomoike Hitonobu
Published: June 2011

Abstract
—The biological actions of tumor necrosis factor-α (TNF-α) are mediated by 2 distinct receptors, TNF-RI (p55) and TNF-RII (p75). The extracellular domains of both receptors are shed in soluble form (sTNF-RI and sTNF-RII). The soluble receptors are involved in regulating TNF-α activities and may have therapeutic potential as TNF-neutralizing agents. However, it remains unclear as to what kind of physiological molecule can regulate TNF receptors. Nitric oxide (NO) mediates a variety of biological and pathophysiological functions. We hypothesized that NO may modulate the expression and shedding of TNF-RI. An NO donor, diethylamine/NO complex (NOC 5), increased sTNF-RI in the supernatants of ECV304, a human umbilical vein cell line, in a dose-dependent manner. TNF-RI mRNA in these cells was upregulated by NOC 5. 8-Br-cGMP and peroxynitrate had no effect on sTNF-RI release. Genistein and herbimycin A, inhibitors of tyrosine kinase, inhibited sTNF-RI release. Herbimycin A inhibited the levels of TNF-RI mRNA enhanced by NOC 5, which downregulated the surface expression of TNF-RI, indicating that NO is also involved in the shedding process of TNF-RI. The shedding of TNF-RI was abolished by a synthetic inhibitor of matrix metalloproteinase, KB-R8301. In conclusion, NO enhanced the release of sTNF-RI from endothelial cells by a cGMP-independent mechanism. Dual pathways suggested for NO-induced sTNF-RI release include (1) enhanced expression of TNF-RI, at least partially, by a tyrosine kinase–dependent mechanism and (2) increased shedding of TNF-RI by a type of metalloproteinase.
Expression of Mouse Acute-Phase (SAA1.1) and Constitutive (SAA4) Serum Amyloid A Isotypes
Authors: Kindy Mark S. , de Beer Maria C. , Yu Jin , de Beer Frederick C.
Published: June 2011

Abstract
—The serum amyloid A (SAA) family of proteins consists of inducible acute-phase members and a constitutive member that are minor apolipoproteins of normal high density lipoprotein (HDL). During inflammation, HDL cholesterol and apolipoprotein A-I (apoA-I) protein are decreased, and these changes are thought to be partly related to the increase in acute-phase SAA proteins that associate with the HDL particle to become the major apolipoprotein species. To determine the specific role of SAA in the alteration of HDL in the absence of a generalized acute-phase response, acute-phase
Saa1.1
transgene expression was directed via an inducible mouse metallothionein promoter. Elevated levels of SAA1.1 (28±9 mg/dL) comparable to a moderate acute-phase response were achieved over a 5-day period. SAA association with the HDL particles at this concentration did not significantly alter the apoA-I or HDL cholesterol levels or change the lipoprotein profiles in the transgenic mice compared with wild-type mice. In addition, we used adenoviral vectors to increase the SAA expression to levels seen in a major acute-phase response. Injection of adenovirus expressing the mouse SAA1.1 protein resulted in high-level expression (72±8 mg/dL) but did not alter apoA-I levels. However, the SAA associated with the HDL particle gave rise to significantly larger HDL particles (≈10%). Adenoviral expression of the constitutive SAA4 protein resulted in an increase in HDL size (≈10%) and an increase in very low density lipoprotein levels (20-fold) and triglyceride levels (1.7-fold). These studies suggest that increases in acute-phase SAA proteins alone are insufficient to alter HDL cholesterol or apoA-I levels during inflammation. A role for constitutive SAA4 in HDL–very low density lipoprotein interactions should be considered.
Quantification In Situ of Crystalline Cholesterol and Calcium Phosphate Hydroxyapatite in Human Atherosclerotic Plaques by Solid-State Magic Angle Spinning NMR
Authors: Guo Wen , Morrisett Joel D. , DeBakey Michael E. , Lawrie Gerald M. , Hamilton James A.
Published: June 2011

Abstract
—Because of renewed interest in the progression, stabilization, and regression of atherosclerotic plaques, it has become important to develop methods for characterizing structural features of plaques in situ and noninvasively. We present a nondestructive method for ex vivo quantification of 2 solid-phase components of plaques: crystalline cholesterol and calcium phosphate salts. Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of human carotid endarterectomy plaques revealed
13
C resonances of crystalline cholesterol monohydrate and a
31
P resonance of calcium phosphate hydroxyapatite (CPH). The spectra were obtained under conditions in which there was little or no interference from other chemical components and were suitable for quantification in situ of the crystalline cholesterol and CPH. Carotid atherosclerotic plaques showed a wide variation in their crystalline cholesterol content. The calculated molar ratio of liquid-crystalline cholesterol to phospholipid ranged from 1.1 to 1.7, demonstrating different capabilities of the phospholipids to reduce crystallization of cholesterol. The spectral properties of the phosphate groups in CPH in carotid plaques were identical to those of CPH in bone.
31
P MAS NMR is a simple, rapid method for quantification of calcium phosphate salts in tissue without extraction and time-consuming chemical analysis. Crystalline phases in intact atherosclerotic plaques (ex vivo) can be quantified accurately by solid-state
13
C and
31
P MAS NMR spectroscopy.
Impact of Antioxidants and HDL on Glycated LDL–Induced Generation of Fibrinolytic Regulators From Vascular Endothelial Cells
Authors: Ren Song , Shen Garry X.
Published: June 2011

Abstract
—Hyperglycemia and dyslipoproteinemia are biochemical markers of diabetes mellitus (DM). Elevated levels of plasminogen activator inhibitor-1 (PAI-1) with and without reduction of tissue plasminogen activator (tPA) in plasma have been frequently found in patients with DM. Our previous studies indicated that glycation enhances low density lipoprotein (LDL)-induced production of PAI-1 and further decreases tPA generation in vascular endothelial cells (ECs). The present study demonstrated that treatment with antioxidants, butylated hydroxytoluene or vitamin E, blocked native LDL– and glycated LDL–induced changes in PAI-1 and tPA generation in ECs. Native or glycated high density lipoprotein (HDL) did not significantly alter tPA generation in ECs. Glycated but not native HDL (≥100 μg/mL) moderately increased PAI-1 release from ECs. Cotreatment with native or glycated HDL inhibited LDL-induced or glycated LDL–induced changes in PAI-1 and tPA generation in ECs. The abundance of conjugated dienes was increased in glycated or EC-modified LDL. Treatment with butylated hydroxytoluene, vitamin E, or HDL reduced the abundance of conjugated dienes in glycated or EC-modified LDL. The effects of antioxidants and HDL on LDL-induced or its glycated LDL–induced changes in the generation of PAI-1 and tPA were also found in cultured human coronary artery ECs. The findings of the present study suggest that antioxidants and HDL may attenuate native LDL– or glycated LDL–induced changes in the generation of fibrinolytic regulators from vascular ECs, which possibly results from their inhibition on the lipid peroxidation of LDL particles. Treatment with antioxidants or hypolipidemic agents potentially improves fibrinolytic activity and reduces thrombotic tendencies in patients with DM.
Oxidation of LDL by Myeloperoxidase and Reactive Nitrogen Species
Authors: Carr Anitra C. , McCall Mark R. , Frei Balz
Published: June 2011

Abstract
—Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. These reactive species oxidize the protein, lipid, and antioxidant components of LDL. Modification of apolipoprotein B results in enhanced uptake of LDL by macrophages with subsequent formation of lipid-laden foam cells. Nitric oxide synthases produce nitric oxide and, under certain conditions, superoxide radicals. Numerous other sources of superoxide radicals have been identified in the arterial wall, including NAD(P)H oxidases and xanthine oxidase. Nitric oxide and superoxide readily combine to form peroxynitrite, a reactive nitrogen species capable of modifying LDL. In this review, we examine the reaction pathways involved in LDL oxidation by myeloperoxidase and reactive nitrogen species and the potential protective effects of the antioxidant vitamins C and E.
Involvement of Aldose Reductase in Vascular Smooth Muscle Cell Growth and Lesion Formation After Arterial Injury
Authors: Ruef Johannes , Liu Si-Qi , Bode Christoph , Tocchi Monica , Srivastava Sanjay , Runge Marschall S. , Bhatnagar Aruni
Published: June 2011

Abstract
—Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy-
trans
-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg · kg
−1
· d
−1
) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.
Increased Production of HDL ApoA-I in Homozygous Familial Defective ApoB-100
Authors: Schaefer J. R. , Winkler K. , Schweer H. , Hoffmann M. M. , Soufi M. , Scharnagl H. , Maisch B. , Wieland H. , Steinmetz A. , März W.
Published: June 2011

Abstract
—Familial defective apolipoprotein (apo) B-100 (FDB) is a frequent cause of hypercholesterolemia. Hypercholesterolemia in homozygous FDB is less severe than in homozygotes for familial hypercholesterolemia. Recently, we showed decreased low density lipoprotein (LDL) apoB-100 fractional catabolism and decreased production of LDL due to an enhanced removal of apoE-containing precursors in a patient with homozygous FDB. The effects of defective apoB-100 on high density lipoprotein (HDL) metabolism are unknown. We studied HDL apoA-I metabolism in this FDB patient and in 6 control subjects by using
2
H
3
-
l
-leucine as a tracer. ApoA-I levels were normal in all study subjects. However, the fractional catabolic rate and the production rate of apoA-I were increased, by 79% and 70%, respectively, in FDB; the fractional catabolic rate of apoA-I in FDB was 0.34 day
−1
compared with 0.19±0.03 day
−1
in normal controls. The production rate of apoA-I in FDB was 18.4 mg · kg
−1
· d
−1
compared with 10.8±2.3 mg · kg
−1
· d
−1
in controls. Thus, we have shown for the first time that defective apoB-100 may influence HDL kinetics. The increase in total HDL turnover might enhance reverse cholesterol transport and could contribute to the seemingly benign clinical course of FDB compared with that of familial hypercholesterolemia.
Effects of ApoE Genotype on ApoB-48 and ApoB-100 Kinetics With Stable Isotopes in Humans
Authors: Welty Francine K. , Lichtenstein Alice H. , Barrett P. Hugh R. , Jenner Jennifer L. , Dolnikowski Gregory G. , Schaefer Ernst J.
Published: June 2011

Abstract
—Subjects with the apolipoprotein (apo) E4 allele have been shown to have higher low density lipoprotein (LDL) cholesterol and apoB levels than do subjects with the other alleles. To elucidate the metabolic mechanisms responsible for this finding, we examined the kinetics of apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL by using a primed constant infusion of [5,5,5-
2
H
3
]leucine in the fed state (hourly feeding) during consumption of an average American diet in 18 normolipidemic subjects, 12 of whom had the apoE3/E3 genotype and 6, the apoE3/E4 genotype. Lipoproteins were isolated by ultracentrifugation and apolipoproteins, by sodium dodecyl sulfate gels; isotope enrichment was assessed by gas chromatography–mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with
saam
II software. Compared with the apoE3/E3 subjects, the apoE3/E4 subjects had significantly higher levels of total apoB, 100.1±17.8 versus 135.4±34.0 mg/dL (
P
=0.009), and significantly higher levels of LDL apoB-100, 88.1±19.2 versus 127.5±32.7 mg/dL (
P
=0.005), respectively. The pool size of TRL apoB-48 was 17.4% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 33.3% lower production rate (
P
=0.28). There was no significant difference in the TRL apoB-48 fractional catabolic rate (5.1±2.2 versus 5.0±2.1 pools per day). The pool size for VLDL apoB-100 was 36% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due entirely to a 30% lower production rate (
P
=0.04). The LDL apoB-100 pool size was 57.8% higher (
P
=0.003) for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 35.5% lower fractional catabolic rate of LDL apoB-100 (
P
=0.003), with no significant difference in production rate. In addition, 77% of VLDL apoB-100 was converted to LDL apoB-100 in apoE3/E4 subjects compared with 58% in apoE3/E3 subjects (
P
=0.05). In conclusion, the presence of 1 E4 allele was associated with higher LDL apoB-100 levels owing to lower fractional catabolism of LDL apoB-100 and a 33% increase in the conversion of VLDL apoB-100 to LDL apoB-100.
MRI Cerebral White Matter Lesions and Paraoxonase
PON1
Polymorphisms
Authors: Schmidt Reinhold , Schmidt Helena , Fazekas Franz , Kapeller Peter , Roob Gudrun , Lechner Anita , Kostner Gert M. , Hartung Hans-Peter
Published: June 2011

Abstract
—White matter lesions (WMLs) on magnetic resonance imaging (MRI) scans of older persons are thought to be caused by cerebral small-vessel disease. As they progress, these brain abnormalities frequently result in cognitive decline and gait disturbances, and their predictors are incompletely understood. Genetic risk factors have been implicated but remain undetermined so far. We examined whether 2 common polymorphisms of the paraoxonase (
PON1
) gene leading to a methionine (
M
allele)–leucine (
L
allele) interchange at position 54 and an arginine (
B
allele)–glutamine (
A
allele) interchange at position 191 are associated with the presence and progression of WMLs. We studied 264 community-dwelling subjects without neuropsychiatric disease (ages 44 to 75 years). All underwent vascular risk factor assessment, brain MRI, and
PON1
genotyping. MRI scanning was repeated after 3 years. The extent and number of WMLs were recorded by 3 independent readers. Progression of WMLs was assessed by direct scan comparison. The final rating relied on the majority judgment of the 3 readers. The
LL
,
LM,
and
MM
genotypes were noted in 111 (42.0%), 118 (44.7%), and 35 (13.3%) subjects, respectively; the
AA,
AB,
and
BB
genotypes occurred in 146 (55.3%), 98 (37.1%), and 20 (7.8%) individuals, respectively. Carriers of the
LL
genotype showed a nonsignificant trend toward more extensive WMLs and more frequently demonstrated lesion progression over the 3-year observation period (
P
=0.03). The polymorphism at position 191 had no effect. Logistic regression analysis yielded age (odds ratio, 1.08/y), diastolic blood pressure (odds ratio, 1.05/mm Hg), and
LL
paraoxonase genotype (odds ratio, 2.65) to be significant predictors of WML progression. These data suggest that the
LL
PON1
genotype at position 54 influences the extent and progression of WMLs in elderly subjects.
Biphasic Effects of the Natural Estrogen 17β-Estradiol on Hepatic Cholesterol Metabolism in Intact Female Rats
Authors: Parini Paolo , Angelin Bo , Stavréus-Evers Anneli , Freyschuss Bo , Eriksson Håkan , Rudling Mats
Published: June 2011

Abstract
—The protective influence of estrogens in cardiovascular disease is believed to be partly due to beneficial effects on cholesterol metabolism. Much of the experimental data are based on models in which synthetic estrogens have been used in pharmacological doses, and therefore, the physiological role of estrogens in cholesterol metabolism is uncertain. To evaluate this important issue, we performed experiments in intact female rats with use of the natural estrogen 17β-estradiol (E2) administered either subcutaneously or orally. After physiological doses of E2 (≤0.04 mg · kg
−1
· d
−1
) were administered, plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I were increased. In the liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7α-hydroxylase activities were increased, as well as cholesterol 7α-hydroxylase mRNA levels. These effects were abolished during treatment with higher doses of E2, whereas apo A-I mRNA increased in a dose-dependent way. After treatment with pharmacological doses of E2 (≥0.2 mg · kg
−1
· d
−1
), the number of hepatic low density lipoprotein receptors increased and plasma cholesterol was reduced. These effects were similar after both oral and subcutaneous administration of E2. Our results show that the responses to E2 are biphasic: plasma HDL, apo A-I, and hepatic enzyme activities governing bile acid and cholesterol synthesis increased only at physiological doses of E2. At pharmacological doses of E2, hepatic low density lipoprotein receptors are stimulated and plasma cholesterol is reduced. Therefore, under physiological conditions, E2 exerts its major effects on hepatic cholesterol metabolism through mechanisms other than stimulation of low density lipoprotein receptor expression.
Periadventitial Inducible Nitric Oxide Synthase Expression and Intimal Thickening
Authors: De Meyer Guido R. Y. , Kockx Mark M. , Cromheeke Kristel M. , Seye Cheikh I. , Herman Arnold G. , Bult Hidde
Published: June 2011

Abstract
—Positioning a silicone collar around the rabbit carotid artery induces a smooth muscle cell–rich intimal thickening. We investigated the localization of inducible nitric oxide synthase (iNOS) during thickening of the intima, the effect of iNOS inhibition on intimal thickness, and the effect of oxidized LDL (ox-LDL) on iNOS expression in the vessel wall. Collars were positioned for 18 hours or for 3, 7, or 14 days. Arterial cross sections were immunostained for iNOS, including naïve, sham-operated, and carotid arteries in which ox-LDL had been infused locally for 14 days. Furthermore, collars were connected to osmotic minipumps for local delivery (5 μL · h
−1
, 14 days, n=12) of saline or the iNOS inhibitor
l
-
N
6
-(1-iminoethyl)-lysine-HCl (L-NIL, 10 mmol/L). In the adventitia and the periadventitial granulation tissue of collared arteries, iNOS-positive macrophages and T lymphocytes were present from 18 hours onward. The media and intima were negative for iNOS. Reverse transcription–polymerase chain reaction revealed iNOS mRNA in collared but not in sham-operated arteries. Local inhibition of iNOS doubled the intimal thickness and decreased nitrotyrosine staining. Ox-LDL–treated arteries, which had a thicker intima, showed a pronounced influx of macrophages and T lymphocytes in all layers of the vessel wall, accompanied by iNOS expression in a subpopulation of these cells. Our study indicates that iNOS was not induced in intimal thickenings predominantly consisting of smooth muscle cells. However, iNOS was expressed in (peri)adventitial tissue and counteracted the progression of intimal thickening. Ox-LDL treatment was accompanied by an abundant influx of iNOS-positive macrophages and T lymphocytes in the vessel, but this could not prevent the progression of intimal thickening.
Low Levels of Extrahepatic Nonmacrophage ApoE Inhibit Atherosclerosis Without Correcting Hypercholesterolemia in ApoE-Deficient Mice
Authors: Thorngate Fayanne E. , Rudel Lawrence L. , Walzem Rosemary L. , Williams David L.
Published: June 2011

Abstract
—The prevention of atherosclerosis by apolipoprotein E (apoE) is generally attributed to the removal of plasma lipoprotein remnant particles. We developed transgenic apoE-knockout mice expressing apoE specifically in the adrenal gland and found that only 3% of the wild-type plasma level of apoE was sufficient to normalize plasma cholesterol levels in the apoE-deficient mouse. As expected, mice expressing apoE at levels that correct hypercholesterolemia had almost no cholesteryl ester deposition in their aortas. In contrast, their nontransgenic siblings had significant atherosclerosis. Unexpectedly, we found that atherosclerosis was also reduced in 2 transgenic lines expressing too little apoE (<1% to 2% of wild type) to correct their hypercholesterolemia. Gel exclusion chromatographic profiles of plasma lipoproteins and the size distributions of lipoproteins with density <1.063 (low density and very low density lipoproteins), as determined by dynamic laser light scattering, were the same in mice expressing <2 μg/mL plasma apoE and their nontransgenic littermates. We conclude that the antiatherogenic action of low levels of plasma apoE is not due to the clearance of remnant lipoproteins. Thus, low levels of apoE provided systemically, but not made in the liver or in macrophages, can block atherogenesis in the vascular wall independently of normalizing the plasma concentration of atherogenic remnant lipoprotein particles.