bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive Oxygen Species Mediate Endothelium-Dependent Relaxations in Tetrahydrobiopterin-Deficient Mice
Authors: Cosentino Francesco , Barker Jane E. , Brand Michael P. , Heales Simon J. , Werner Ernst R. , Tippins John R. , West Nick , Channon Keith M. , Volpe Massimo , Lüscher Thomas F.
Published: June 2011

Abstract
—(6
R
)-5,6,7,8-Tetrahydro-biopterin (H
4
B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H
4
B synthesis. A relative shortage of H
4
B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H
4
B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BL×CBA) were compared. H
4
B levels were determined by high-performance liquid chromatography and NO synthase activity by [
3
H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H
4
B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with
N
G
-nitro-
l
-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H
4
B, the differences between the 2 groups disappeared. Our findings demonstrate that H
4
B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H
4
B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.
Myosin Light Chain Kinase Regulates Capacitative Ca
2+
Entry in Human Monocytes/Macrophages
Authors: Tran Quang-Kim , Watanabe Hiroshi , Le Hong-Yen , Pan Ling , Seto Minoru , Takeuchi Kazuhiko , Ohashi Kyoichi
Published: June 2011

Abstract
—Monocytes/macrophages are present in all stages of atherosclerosis. Although many of their activities depend to various extents on changes in intracellular Ca
2+
concentration ([Ca
2+
]
i
), mechanisms regulating [Ca
2+
]
i
in these cells remain unclear. We aimed to explore the role of myosin light chain kinase (MLCK) in Ca
2+
signaling in freshly isolated human monocytes/macrophages. Large capacitative Ca
2+
entry (CCE) was observed under fura 2 fluoroscopy in human monocytes/macrophages treated with thapsigargin and cyclopiazonic acid. ML-9 and wortmannin, 2 structurally different inhibitors of MLCK, dose-dependently (1 to 100 μmol/L) prevented CCE and completely did so at 100 μmol/L, whereas inhibitors of tyrosine kinase and protein kinase C had only partial effects. Western blotting showed that thapsigargin significantly caused myosin light chain phosphorylation, which was almost completely blocked by ML-9 (100 μmol/L) and wortmannin (100 μmol/L). ML-9 also dose-dependently (1 to 100 μmol/L) inhibited this phosphorylation, which was well correlated with its inhibition of CCE. Transfection with MLCK antisense completely prevented CCE in response to thapsigargin and cyclopiazonic acid, whereas MLCK sense had no effect. These data strongly indicate that MLCK regulates CCE in human monocytes/macrophages. The study suggests a possible involvement of MLCK in many Ca
2+
-dependent activities of monocytes/macrophages.
Role of Sex Differences and Effects of Endothelial NO Synthase Deficiency in Responses of Carotid Arteries to Serotonin
Authors: Lamping Kathryn G. , Faraci Frank M.
Published: June 2011

Abstract
—We examined the hypothesis that contraction of the carotid arteries to serotonin is normally inhibited by endothelial NO synthase (eNOS) and is enhanced in mice lacking the gene for eNOS. Because the influence of eNOS may vary with the sex of the mouse, we also tested whether responses to serotonin were dependent on sex. We studied carotid arteries in vitro from littermate control (eNOS
+/+
) mice, heterozygous (eNOS
+/−
) mice, and homozygous eNOS-deficient (eNOS
−/−
) mice (male and female). Contraction to serotonin was greater in male eNOS
+/+
mice than in female eNOS
+/+
mice. In male mice, contraction to serotonin increased by ≈40% and 2.5-fold in male eNOS
+/−
and eNOS
−/−
mice, respectively. Contraction to serotonin was more than doubled in female eNOS
+/−
mice and increased >5-fold in arteries from eNOS
−/−
mice. In contrast, maximum vasoconstriction to U46619 was similar in male and female eNOS
+/+
, eNOS
+/−
, and eNOS
−/−
mice. Relaxation to acetylcholine was not different in male and female eNOS
+/+
or eNOS
+/−
mice but was absent in eNOS
−/−
mice. These findings suggest that the contraction of carotid arteries to serotonin is influenced by the sex of the animal. eNOS deficiency in gene-targeted mice is associated with enhanced contraction to serotonin, particularly in female mice, providing direct evidence that eNOS is a major determinant of vascular effects of serotonin. The results with eNOS
+/−
mice suggest a “gene-dosing” effect for vascular responses to serotonin.
Human Paraoxonase-3 Is an HDL-Associated Enzyme With Biological Activity Similar to Paraoxonase-1 Protein but Is Not Regulated by Oxidized Lipids
Authors: Reddy Srinivasa T. , Wadleigh David J. , Grijalva Victor , Ng Carey , Hama Susan , Gangopadhyay Aditya , Shih Diana M. , Lusis Aldons J. , Navab Mohamad , Fogelman Alan M.
Published: June 2011

Abstract
—Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL) and participates in the prevention of low density lipoprotein (LDL) oxidation. Two other paraoxonase (PON) family members, namely, PON2 and PON3, have been identified. In this study, we report the cloning and characterization of the human PON3 gene from HepG2 cells. Tissue Northern analysis identifies an ≈1.3-kb transcript for PON3 primarily in the liver. PON3-specific peptide antibodies detect an ≈40-kDa protein associated with HDL and absent from LDL. Pretreatment of cultured human aortic endothelial cells with supernatants from HeLa Tet On cell lines overexpressing PON3 prevents the formation of mildly oxidized LDL and inactivates preformed mildly oxidized LDL. In contrast to PON1, PON3 is not active against the synthetic substrates paraoxon and phenylacetate. Furthermore, PON3 expression is not regulated in HepG2 cells by oxidized phospholipids and is not regulated in the livers of mice fed a high-fat atherogenic diet.
Oxidized LDL Regulates Vascular Endothelial Growth Factor Expression in Human Macrophages and Endothelial Cells Through Activation of Peroxisome Proliferator–Activated Receptor-γ
Authors: Inoue Mayumi , Itoh Hiroshi , Tanaka Tokuji , Chun Tae-Hwa , Doi Kentaro , Fukunaga Yasutomo , Sawada Naoki , Yamshita Jun , Masatsugu Ken , Saito Takatoshi , Sakaguchi Satsuki , Sone Masakatsu , Yamahara Ken-ichi , Yurugi Takami , Nakao Kazuwa
Published: June 2011

Abstract
—Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator–activated receptor-γ (PPARγ) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPARγ may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-(
S
)-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPARγ ligands. Therefore, we investigated the involvement of PPARγ in the regulation of VEGF by Ox-LDL. PPARγ expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 μg/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPARγ activators, troglitazone (TRO), and 15-deoxy-Δ
12,14
-prostaglandin J
2
(PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPARγ expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPARγ (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPARγ.
Dietary Cosupplementation With Vitamin E and Coenzyme Q
10
Inhibits Atherosclerosis in Apolipoprotein E Gene Knockout Mice
Authors: Thomas Shane R. , Leichtweis Steven B. , Pettersson Knut , Croft Kevin D. , Mori Trevor A. , Brown Andrew J. , Stocker Roland
Published: June 2011

Abstract
—Intimal oxidation of LDL is considered an important early event in atherogenesis, and certain antioxidants are antiatherogenic. Dietary coenrichment with vitamin E (VitE) plus ubiquinone-10 (CoQ
10
, which is reduced during intestinal uptake to the antioxidant ubiquinol-10, CoQ
10
H
2
) protects, whereas enrichment with VitE alone can increase oxidizability of LDL lipid against ex vivo oxidation. In the present study, we tested whether VitE plus CoQ
10
cosupplementation is more antiatherogenic than either antioxidant alone, by use of apolipoprotein E–deficient (apoE−/−) mice fed a high-fat diet without (control) or with 0.2% (wt/wt) VitE, 0.5% CoQ
10
, or 0.2% VitE plus 0.5% CoQ
10
(VitE+CoQ
10
) for 24 weeks. None of the supplements affected plasma cholesterol concentrations, whereas in the VitE and CoQ
10
groups, plasma level of the respective supplement increased. Compared with control, plasma from CoQ
10
or VitE+CoQ
10
but not VitE-supplemented animals was more resistant to ex vivo lipid peroxidation induced by peroxyl radicals. VitE supplementation increased VitE levels in aorta, heart, brain, and skeletal muscle, whereas CoQ
10
supplementation increased CoQ
10
only in plasma and aorta and lowered tissue VitE. All treatments significantly lowered aortic cholesterol compared with control, but only VitE+CoQ
10
supplementation significantly decreased tissue lipid hydroperoxides when expressed per parent lipid. In contrast, none of the treatments affected aortic ratios of 7-ketocholesterol to cholesterol. Compared with controls, VitE+CoQ
10
supplementation decreased atherosclerosis at the aortic root and arch and descending thoracic aorta to an extent that increased with increasing distance from the aortic root. CoQ
10
significantly inhibited atherosclerosis at aortic root and arch, whereas VitE decreased disease at aortic root only. Thus, in apoE−/− mice, VitE+CoQ
10
supplements are more antiatherogenic than CoQ
10
or VitE supplements alone and disease inhibition is associated with a decrease in aortic lipid hydroperoxides but not 7-ketocholesterol.
Platelet Adhesion Enhances the Glycoprotein VI–Dependent Procoagulant Response
Authors: Siljander Pia , Farndale Richard W. , Feijge Marion A. H. , Comfurius Paul , Kos Snjezana , Bevers Edouard M. , Heemskerk Johan W. M.
Published: June 2011

Abstract
—In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca
2+
signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca
2+
]
i
. Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.
Increased NAD(P)H Oxidase and Reactive Oxygen Species in Coronary Arteries After Balloon Injury
Authors: Shi Yi , Niculescu Rodica , Wang Dian , Patel Sachin , Davenpeck Kelly L. , Zalewski Andrew
Published: June 2011

Abstract
—Reactive oxygen species (ROS), produced by cellular constituents of the arterial wall, provide a signaling mechanism involved in vascular remodeling. Because adventitial fibroblasts are actively involved in coronary remodeling, we examined whether the changes in the redox state affect their phenotypic characteristics. To this end, superoxide anion production and NAD(P)H oxidase activity were measured in porcine coronary arteries in vivo, and the effect of ROS generation on adventitial fibroblast proliferation was examined in vitro. Superoxide production (SOD- and Tiron-inhibitable nitro blue tetrazolium [NBT] reduction) increased significantly within 24 hours after balloon-induced injury, with the product of NBT reduction present predominantly in adventitial fibroblasts. These changes were NAD(P)H oxidase–dependent, because diphenyleneiodonium (DPI) abolished superoxide generation (
P
<0.001). Furthermore, the injury-induced superoxide production was associated with augmented NAD(P)H oxidase activity and upregulation of p47
phox
and p67
phox
in adventitial fibroblasts (immunohistochemistry). Serum stimulation of isolated adventitial fibroblasts produced time-dependent increases in ROS production (peak 3 to 6 hours). The inhibition of ROS generation with NAD(P)H oxidase inhibitor (DPI) or the removal of ROS with antioxidants (Tiron, catalase) abrogated proliferation of adventitial fibroblasts. These results indicate that vascular NAD(P)H oxidase plays a central role in the upregulation of oxidative stress after coronary injury, providing pivotal growth signals for coronary fibroblasts.
Adenosine A
2A
Receptor Stimulation Reduces Inflammation and Neointimal Growth in a Murine Carotid Ligation Model
Authors: McPherson John A. , Barringhaus Kurt G. , Bishop Gregory G. , Sanders John M. , Rieger Jayson M. , Hesselbacher Sean E. , Gimple Lawrence W. , Powers Eric R. , Macdonald Timothy , Sullivan Gail , Linden Joel , Sarembock Ian J.
Published: June 2011

Abstract
—Endothelial activation and leukocyte recruitment are early events in atherosclerosis and the vascular response to injury. Adenosine has anti-inflammatory effects on leukocytes and endothelial cells mediated through its A
2A
receptor. We tested the hypothesis that A
2A
activation would reduce inflammation and neointimal formation in a murine carotid ligation model. Before injury, mice were randomized to a 7-day subcutaneous infusion of a specific A
2A
receptor agonist (ATL-146e, 0.004 μg/kg per minute), vehicle control, ATL-146e plus ZM241385 (a selective A
2A
antagonist), or ZM241385 alone. Leukocyte recruitment and adhesion molecule expression were assessed at early time points, and the neointimal area was measured at 14 and 28 days after injury. Compared with control mice, ATL-146e–treated mice had significantly less neutrophil and macrophage recruitment and vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and P-selectin expression in the first 7 days after injury. Neointimal area was markedly and persistently reduced by 80% at 14 and 28 days, despite termination of ATL infusion at 7 days. ATL-146e+ZM241385–treated and ZM241385-treated animals had neointimal areas similar to those of control animals, confirming that the observed effects of ATL-146e were mediated specifically by the A
2A
receptor. These data demonstrate that novel stimulation of adenosine A
2A
receptors can inhibit early inflammatory processes that are important in neointimal formation after vascular injury.
cAMP Signal Transduction Cascade, a Novel Pathway for the Regulation of Endothelial Nitric Oxide Production in Coronary Blood Vessels
Authors: Zhang Xiaoping , Hintze Thomas H.
Published: June 2011

Abstract
—The aim of this study was to determine whether cAMP signal transduction plays a role in the regulation of endothelial nitric oxide (NO) production. Canine coronary blood vessels were isolated, and nitrite, the hydration product of NO, from these vessels was quantified by using the Griess reaction. Forskolin (10
−4
mol/L), 8-bromo-cAMP (10
−2
mol/L), or isoproterenol (10
−4
mol/L) significantly increased nitrite release to 168±10, 162±13, or 149±13 pmol/mg, respectively, from isolated coronary microvessels (all
P
<0.05; control, 86±3 pmol/mg). Adrenomedullin and calcitonin gene–related peptide (CGRP), both potent vasodilator peptides, also increased coronary microvascular nitrite production.
N

ω

-nitro-
l
-arginine methyl ester, a competitive inhibitor of NO synthase, or Rp-cAMP, a protein kinase A inhibitor, markedly blocked the nitrite release induced by these agents. Forskolin and adrenomedullin also potentiated coronary NO production induced by bradykinin. In large coronary arteries, removal of the endothelium eliminated nitrite production to both forskolin and acetylcholine. Our data demonstrate that stimulation of cAMP signal transduction can substantially increase coronary NO production, indicating that there is a cAMP-mediated, endothelial NO–forming system in coronary blood vessels. Because the cAMP signal cascade can be activated by CGRP or adrenomedullin and enhance kinin-mediated nitrite production, the cAMP-NO pathway may play an important role in the regulation of cardiovascular function.
Bisphosphonates Alendronate and Ibandronate Inhibit Artery Calcification at Doses Comparable to Those That Inhibit Bone Resorption
Authors: Price Paul A. , Faus Samuel A. , Williamson Matthew K.
Published: June 2011

Abstract
—The present experiments were carried out to test the hypothesis that artery calcification is linked to bone resorption by determining whether the selective inhibition of bone resorption with the bisphosphonates alendronate and ibandronate will inhibit artery calcification. Artery calcification was first induced by treatment of 42-day-old male rats with warfarin, a procedure that inhibits the γ-carboxylation of matrix Gla protein and has been shown to cause extensive calcification of the artery media within 2 weeks. These experiments revealed that ibandronate (0.05 mg · kg
−1
· d
−1
) and alendronate (0.1 mg · kg
−1
· d
−1
) completely inhibited calcification of all arteries and heart valves examined after 2 and 4 weeks of warfarin treatment. A 10-fold lower dose of alendronate reduced artery calcification by 50% (
P
<0.005). These bisphosphonate doses are comparable to those that inhibit bone resorption in rats of this age. More rapid artery calcification was induced by treatment with warfarin together with high doses of vitamin D, a procedure that causes extensive artery calcification by 84 hours. Alendronate and ibandronate again completely inhibited calcification of all arteries and heart valves examined. The subcutaneous doses of alendronate and ibandronate necessary to inhibit artery calcification are comparable to the daily subcutaneous doses of these drugs that have previously been shown to inhibit bone resorption in rats of the same age, with 50% inhibition of artery calcification at 20 μg alendronate · kg
−1
· d
−1
and at 1 μg ibandronate · kg
−1
· d
−1
. Bisphosphonate treatment did not affect serum calcium and phosphate, and so the inhibition of artery calcification cannot be due to a simple lowering of the serum calcium phosphate ion product. We conclude that bisphosphonates inhibit the calcification of arteries and heart valves at doses comparable to the doses that inhibit bone resorption. These results support the hypothesis that artery calcification is linked to bone resorption. The mechanism of this linkage remains to be established, however, and an alternative explanation for the present results is also considered.
Renal Insufficiency, Vitamin B
12
Status, and Population Attributable Risk for Mild Hyperhomocysteinemia Among Coronary Artery Disease Patients in the Era of Folic Acid–Fortified Cereal Grain Flour
Authors: Liaugaudas Gintaras , Jacques Paul F. , Selhub Jacob , Rosenberg Irwin H. , Bostom Andrew G.
Published: June 2011

Abstract
—Fortification of enriched cereal grain flour products with folic acid has drastically reduced the prevalence of deficient plasma folate status, a major determinant of plasma total homocysteine (tHcy) levels. We hypothesized that even more liberally defined “suboptimal” plasma folate status might no longer contribute importantly to the population attributable risk (PAR) for mild hyperhomocysteinemia, a putative atherothrombotic risk factor. We determined fasting plasma tHcy, folate, vitamin B
12
, and pyridoxal 5′-phosphate levels, along with serum creatinine and albumin levels, in 267 consecutive patients (aged 61±9 [mean±SD] years, 76.4% men and 26.6% women) with stable coronary artery disease (CAD) who were nonusers of vitamin supplements or had abstained from supplement use for at least 6 weeks before examination. Subjects were evaluated a minimum of 3 months after the implementation of flour fortification was largely completed. Relative risk estimates for the calculation of PAR were derived from a multivariable-adjusted logistic regression model with ≥12 μmol/L tHcy as the dependent variable and with age, sex, pyridoxal 5′-phosphate (continuous), albumin (continuous), <5 ng/mL folate, <250 pg/mL vitamin B
12
, and ≥1.3 mg/dL creatinine as the independent variables. The prevalence of ≥12 μmol/L plasma tHcy was 11.2% (30 of 267 patients). PAR estimates (percentage) for ≥12 μmol/L tHcy were as follows: <5 ng/mL folate (<1%), <250 pg/mL vitamin B
12
(24.5%), and ≥1.3 mg/dL creatinine (37.5%). In the era of folic acid–fortified cereal grain flour, renal insufficiency and suboptimal vitamin B
12
status (but not folate status) contribute importantly to the PAR for mild hyperhomocysteinemia among patients with stable CAD.
Pro-Oxidant Effect of Vitamin E in Cigarette Smokers Consuming a High Polyunsaturated Fat Diet
Authors: Weinberg Richard B. , VanderWerken Barbara S. , Anderson Rachel A. , Stegner Jane E. , Thomas Michael J.
Published: June 2011

Abstract
—Dietary polyunsaturated fats and vitamin E are associated with reduced risk for atherosclerosis, but in smokers, they could promote lipid oxidation. Therefore, we examined the effects of a high polyunsaturated fat diet and vitamin E supplementation on measures of lipid oxidation in cigarette smokers. Ten subjects who smoked >1 pack of cigarettes per day were sequentially fed the following: a baseline diet in which the major fat source was olive oil, a diet in which the major fat source was high-linoleic safflower oil, and finally, the safflower oil diet plus 800 IU vitamin E per day. LDL oxidation lag time and rate and plasma total F
2
-isoprostanes and prostaglandin F
2α
(PGF
2α
) were determined after 3 weeks on each diet. The safflower oil diet increased total F
2
-isoprostanes from 53.0±7.2 to 116.2±11.2 nmol/L and PGF
2α
from 3.5±0.2 to 5.5±0.5 nmol/L, without changing LDL oxidation parameters. Addition of vitamin E prolonged mean LDL oxidation lag time but, paradoxically, further increased F
2
-isoprostanes to 188.2±10.9 nmol/L and PGF
2α
to 7.8±0.4 nmol/L. These data suggest that vitamin E may function as a pro-oxidant in cigarette smokers consuming a high polyunsaturated fat diet.
LDL Modified by Hypochlorous Acid Is a Potent Inhibitor of Lecithin-Cholesterol Acyltransferase Activity
Authors: McCall Mark R. , Carr Anitra C. , Forte Trudy M. , Frei Balz
Published: June 2011

Abstract
—Modification of low density lipoprotein (LDL) by myeloperoxidase-generated HOCl has been implicated in human atherosclerosis. Incubation of LDL with HOCl generates several reactive intermediates, primarily
N
-chloramines, which may react with other biomolecules. In this study, we investigated the effects of HOCl-modified LDL on the activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for high density lipoprotein maturation and the antiatherogenic reverse cholesterol transport pathway. We exposed human LDL (0.5 mg protein/mL) to physiological concentrations of HOCl (25 to 200 μmol/L) and characterized the resulting LDL modifications to apolipoprotein B and lipids; the modified LDL was subsequently incubated with apolipoprotein B–depleted plasma (density >1.063 g/mL fraction), which contains functional LCAT. Increasing concentrations of HOCl caused various modifications to LDL, primarily, loss of lysine residues and increases in
N
-chloramines and electrophoretic mobility, whereas lipid hydroperoxides were only minor products. LCAT activity was extremely sensitive to HOCl-modified LDL and was reduced by 23% and 93% by LDL preincubated with 25 and 100 μmol/L HOCl, respectively. Addition of 200 μmol/L ascorbate or
N
-acetyl derivatives of cysteine or methionine completely prevented LCAT inactivation by LDL preincubated with ≤200 μmol/L HOCl. Protecting the free thiol groups of LCAT with 5,5′-dithio-bis-(2-nitrobenzoic acid) before exposure to HOCl-modified LDL, which inhibits lipid hydroperoxide–mediated inactivation of LCAT, failed to prevent the loss of enzyme activity. Our data indicate that
N
-chloramines from HOCl-modified LDL mediate the loss of plasma LCAT activity and provide a novel mechanism by which myeloperoxidase-generated HOCl may promote atherogenesis.
Lipolysis of LDL by Human Secretory Phospholipase A
2
Induces Particle Fusion and Enhances the Retention of LDL to Human Aortic Proteoglycans
Authors: Hakala Jukka K. , Öörni Katariina , Pentikäinen Markku O. , Hurt-Camejo Eva , Kovanen Petri T.
Published: June 2011

Abstract
—The first morphological sign of atherogenesis is the accumulation of extracellular lipid droplets in the proteoglycan-rich subendothelial layer of the arterial intima. Secretory nonpancreatic phospholipase A
2
(snpPLA
2
), an enzyme capable of lipolyzing LDL particles, is found in the arterial extracellular matrix and in contact with the extracellular lipid droplets. We have recently shown that in the presence of heparin, lipolysis of LDL with bee venom PLA
2
induces aggregation and fusion of the particles. Here, we studied the effect of human snpPLA
2
on the integrity of LDL particles and on their interaction with human aortic proteoglycans. In addition, the capacity of the proteoglycans to retain PLA
2
-lipolyzed LDL particles was tested in a microtiter well assay. We found that lipolysis of LDL induced fusion of proteoglycan-bound LDL particles, which increased their binding strength to the proteoglycans. Moreover, lipolysis of LDL with snpPLA
2
under physiological salt and albumin concentrations induced a 3-fold increase in the amount of LDL bound to proteoglycans. The results imply a role for PLA
2
in the retention and accumulation of LDL to the proteoglycan matrix in atherosclerosis.
Small GTP-Binding Protein Ral Modulates Regulated Exocytosis of von Willebrand Factor by Endothelial Cells
Authors: de Leeuw Hubert P. J. C. , Fernandez-Borja Mar , Reits Eric A. J. , Romani de Wit Thalia , Wijers-Koster Pauline M. , Hordijk Peter L. , Neefjes Jacques , van Mourik Jan A. , Voorberg Jan
Published: June 2011

Abstract
—Weibel-Palade bodies are endothelial cell–specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.
Arachidonic acid availability and prostacyclin production by cultured human endothelial cells.
Authors: Spector A A , Kaduce T L , Hoak J C , Czervionke R L
Published: June 2011

When human umbilical vein endothelial cultures were grown in the presence of supplemental arachidonic acid, the cell phospholipids became enriched with arachidonic acid. Prostacyclin (PGI2) accumulated in the medium during supplementation with arachidonic acid. The capacity of these enriched cultures to produce PGI2 when subsequently incubated with either arachidonic acid or thrombin was reduced by as much as 90%, but release of arachidonic acid from the cell lipids in response to thrombin stimulation was not inhibited. Refractory cultures completely recovered the capacity to form PGI2 within 18 hours after removal of the medium containing supplemental arachidonic acid. However, recovery was prevented by cycloheximide. When enrichment with arachidonic acid was done in the presence of ibuprofen, a reversible cyclooxygenase inhibitor, PGI2 did not accumulate in the medium during supplementation, and the subsequent capacity of the cultures to produce PGI2 in response to thrombin increased by 70% to 240%. By contrast, the capacity of these supplemented cultures to convert added arachidonic acid to PGI2 did not increase. Therefore, the enhancement in thrombin-stimulated PGI2 production when the cultures are supplemented with arachidonic acid probably is due to the larger amount of arachidonic acid available in the intracellular lipid substrate pools, rather than to an activation of the PGI2 synthetic pathway. These findings suggest that changes in the arachidonic acid content of the endothelial cell lipids may modulate the capacity of the endothelium to produce PGI2 in response to stimulation.
Influence of plasma lipoproteins on platelet aggregation in a normal male population.
Authors: Hassall D G , Forrest L A , Bruckdorfer K R , Marenah C B , Turner P , Cortese C , Miller N E , Lewis B
Published: June 2011

Aggregation tests were performed on platelet-rich plasma from healthy male volunteers to determine the minimum concentration of adenosine diphosphate (ADP), epinephrine, collagen, or thrombin required to induce secondary aggregation. Platelets were also analyzed for cholesterol and phospholipid, and in some cases their membrane fluidity was determined by fluorescence depolarization of the probe, diphenylhexatriene. Concentrations of the major lipoprotein fractions in the plasma were measured and related to the sensitivity of platelets to the four agonists. Low density lipoprotein (LDL) and total cholesterol concentrations, but not high density lipoproteins (HDL) or very low density lipoproteins (VLDL), were positively correlated with sensitivity to aggregation by epinephrine, but not by other agonists. By arrangement of the lipoprotein concentration into quintiles, the effect of LDL was most striking in the lower two quintiles, where the sensitivity to adrenaline and ADP were much diminished. The middle and upper two quintiles showed a similar sensitivity. Lower platelet cholesterol/phospholipid ratios were also associated with a reduced sensitivity to epinephrine or ADP, but only at the lower end of the range. Membrane microviscosity was correlated negatively with collagen sensitivity and with VLDL cholesterol concentrations, but positively with HDL cholesterol concentrations. Platelet behavior appears, therefore, to be influenced by lipoprotein concentrations within the range found in a healthy population.
Small liver fenestrae may explain the susceptibility of rabbits to atherosclerosis.
Authors: Wright P L , Smith K F , Day W A , Fraser R
Published: June 2011

Rabbits fed cholesterol rapidly develop high serum cholesterol levels which lead to the development of atherosclerosis. This is related to the retention of cholesterol-rich chylomicron remnants in the circulation. In most animals, such as rats, chylomicron remnants are rapidly removed from the circulation by the liver. The first barrier to this removal is the fenestrated endothelium of liver sinusoids. Measurements made of a large number of sinusoidal fenestrae by scanning electron microscopy have shown the average diameters to be 89 nm in rats and 49 nm in rabbits. We postulate that the small size of endothelial fenestrae in the liver sinusoids of rabbits hinders the egress of chylomicron remnants from the sinusoidal blood, explaining the subsequent development of hypercholesterolemia and atherosclerosis.
Lipid composition of rabbit aortic wall following removal of endothelium by balloon catheter.
Authors: Alavi M , Dunnett C W , Moore S
Published: June 2011

To determine the lipid content of the neointima formed following balloon catheter deendothelialization, we measured the chemical composition of intimal medial samples of the aortic wall of rabbits at 6, 12, and 24 months after the operation. The areas of endothelial regrowth and the denuded areas were outlined by intravenous injection of Evan's blue dye. Lipids were extracted by chloroform/methanol (2:1, vol/vol) separated by thin-layer chromatography, eluted, and estimated by gas-liquid chromatography. No tendency to regression was observed. By contrast we observed a progressive increase over time in the concentration of most lipid classes in the areas of endothelial regrowth. There is evidence indicating that the increase in the lipid concentration parallels the increase in the glycosaminoglycan content in the areas of endothelial regrowth. It is possible that endothelial injury causes some irreversible changes in the composition of the neointima in areas of endothelial regeneration.
Inhibition of LDL degradation in cultured human fibroblasts induced by endothelial cell-conditioned medium.
Authors: Cornicelli J A , Witte L D , Goodman D S
Published: June 2011

Cultured porcine aortic endothelial cells (EC) secrete a potent mitogen into their serum-free culture medium which stimulates DNA synthesis in quiescent human fibroblasts. Previous work has shown that endothelial cell-conditioned medium (ECCM) also stimulates parallel, concentration-dependent increases in low density lipoprotein (LDL) cell surface binding and internalization. LDL degradation, however, was inhibited in ECCM-treated cells. Studies were conducted to determine whether the EC-derived growth factor (EDGF) and the endothelial-derived inhibitor of LDL degradation (EDID) are separate and distinct components, and to further delineate some of the characteristics of EDID. Time-course studies showed that the mitogenic activity released by EC into ECCM increased steadily during the first 24 hours and came to a plateau at about 48 hours of conditioning, whereas inhibitory activity increased linearly in ECCM for at least 6 days. Mitogenic activity in ECCM was stable under the same conditions. Ultrafiltration experiments showed that EDGF activity has an apparent molecular weight in excess of 30,000; in contrast EDID activity was not retained by filters with a 2000 dalton retention limit. EDID activity also failed to bind to a Blue Sepharose affinity column, while EDGF was bound and could be eluted with 1M NaCl in the presence of ethylene glycol. The ammonium ion concentration in ECCM was measured to determine the possible contribution of this ion to EDID activity. Confluent cultures of porcine aortic EC in serum-free culture medium produced substantial amounts of NH4+. After 96 hours of conditioning, the level of NH4+ in ECCM rose from near zero to 2-4 mM. When equivalent amounts of NH4Cl were added to fresh nonconditioned medium, a level of inhibitory (EDID) activity similar to that found with ECCM was observed. These studies indicate that EDGF and EDID activities are distinct and separate components of ECCM. Since NH4+ ion is present in ECCM in concentrations that are sufficient to account for all the observed EDID activity, it is likely that NH4+ is responsible for this activity.
Flow separation in the renal arteries.
Authors: Sabbah H N , Hawkins E T , Stein P D
Published: June 2011

To determine the nature of the flow in the proximal portion of the renal arteries, a common site for the development of atherosclerosis, we performed pulsatile flow studies in a clear acrylic mold of a normal human aorta and renal arteries. The mold was developed from a cast of the renal arteries prepared in situ during the autopsy of a 27-year-old woman. Flow in the mold was visualized by the illumination of bouyant particles (100 to 300 mu). A range of branch (renal) to trunk (aorta) flow ratios of 0.053 to 0.350 was studied. Flow separation during systole was considered present when particles near the wall reversed direction. Flow separation occurred throughout systole at the superior aspect of the origin of both the left and right renal arteries at a branch-to-trunk flow ratio of 0.053. As the branch-to-trunk flow ratio increased, flow separation occurred during the deceleration phase of right renal flow. At a branch-to-trunk flow ratio of 0.350, flow separation was no longer present. This suggests that flow separation may occur near the renal ostia if renal flow falls appreciably while aortic flow remains elevated. Because flow separation involves low wall shear, mass transfer across the arterial wall may be adversely affected and possibly contribute to the formation of atheroma.
Turnover of low density lipoproteins during inhibition of cholesterol absorption by neomycin.
Authors: Kesäniemi Y A , Grundy S M
Published: June 2011

The mechanisms for the hypocholesterolemic actin of neomycin were examined in six patients with various levels of plasma total cholesterol and triglycerides. All patients were studied on a metabolic ward. The first period of 6 weeks was for control. Thereafter, neomycin (1.5 g/day) was started, and the patients were readmitted for another 6-week period after 2 to 3 months of treatment with the drug. Cholesterol balance studies showed that neomycin increased fecal excretion of neutral steroids by an average of 45%; the drug also inhibited absorption of exogenous cholesterol by an average of 44%. During treatment with neomycin, the plasma total cholesterol fell by an average of 20%, low density lipoproteins (LDL) fell by 25%, and high density lipoproteins, by 16%. Neomycin did not change plasma triglyceride levels. Turnover of the apoprotein of LDL (apoLDL) was measured following injection of 125I-apoLDL. Neomycin decreased synthesis of apoLDL by 28%. The decrease in plasma apoLDL level was correlated positively with the decrease in apoLDL synthetic rate. The effect of the drug on clearance of LDL was less constant; four of six patients had an increase in fractional clearance rates of apoLDL, but the change for the whole group was not statistically significant. These data suggest that a decrease in production of LDL is a major factor in the lowering of LDL following inhibition of cholesterol absorption; however, an increase in clearance rates may occur in some patients.
Isolation and characterization of human plasma lipid transfer proteins.
Authors: Albers J J , Tollefson J H , Chen C H , Steinmetz A
Published: June 2011

A highly purified protein that facilitates the exchange and net mass transfer of cholesteryl ester (CE), and triacylglycerol (TG), and the transfer of phosphatidylcholine (PC), between plasma lipoproteins was isolated from the d-1.21-1.25 g/ml plasma fraction. Transfer activities showed similar distributions through ultracentrifugation, phenyl-Sepharose, DEAE-Sepharose, CM-cellulose, and hydroxyapatite chromatography. The lipid transfer protein appears to be an acidic protein with an apparent molecular weight of 64,000 +/- 1600 (n = 4) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent molecular weight of 65,000 by gel filtration chromatography, and has a pI of 5.0 +/- 0.2 (n = 5) by both analytical isoelectricfocusing and chromatofocusing. The purified transfer protein facilitated the net mass transfer of CE from high density lipoprotein (HDL) or low density lipoprotein (LDL) to very low density lipoprotein (VLDL) and the net mass transfer of TG from VLDL to LDL or HDL. Chromatography of lipid transfer protein fractions on a heparin-Sepharose column yielded two separate fractions with PC transfer activity. The first fraction did not bind to heparin column, was relatively resistant to elevated temperature (at 58 degrees C, only 5% activity was lost in 1 hour) and eluted with the CE and TG transfer activities which were also temperature-resistant. The second PC transfer activity bound to the heparin column and was temperature-sensitive (at 58 degrees C, 90% activity was lost in 1 hour). Addition of the temperature-resistant lipid transfer fraction (LTP-1) and purified lecithin cholesterol acyltransferase (LCAT) to whole plasma stimulated the endogenous plasma cholesterol esterification rate by approximately 50%, whereas addition of either LTP-1 or LCAT only slightly enhanced the esterification rate. The transfer of CE, TG, and PC was mediated by a temperature-resistant plasma protein or proteins with very similar properties. Plasma also contained a distinct lipid transfer protein which was temperature-sensitive and facilitated the transfer of PC, but not CE or TG.
Dietary fiber and lipoprotein metabolism in the genetically obese Zucker rat.
Authors: Wilson J N , Wilson S P , Eaton R P
Published: June 2011

The present investigation was designed to examine the influence of dietary fibers with differing soluble fiber compositions upon the metabolism of lipids in a hyperlipemic animal model, the Zucker fatty rat. The response to fiber was examined using a diet supplemented with cellulose, oat bran, or pectin which have a soluble/insoluble fiber ratio of 0:100, 33:66, and 100:0, respectively. These fibers provided 10% of the total diet weight; the control diet contained no fiber. A rapid increase in plasma triglyceride concentration was observed in all animals given fiber-supplemented diets in correlation with the increased carbohydrate content of the defined diets relative to the prestudy diets. This increase in plasma triglyceride was due to increased production of triglycerides with no change in the rates of clearance. The plasma total cholesterol levels were relatively constant on all diets. However, after 7 weeks on the pectin-supplemented diet, rats showed a 39% elevation in HDL and a 44% reduction in LDL concentration. This diet also resulted in reduced weight gain, in spite of a caloric intake equivalent to the control diets. Our data suggest that the ability of dietary fiber to alter plasma lipoproteins might be predictable from the soluble fiber composition or the pectin content of a given dietary fiber in this model of genetic endogenous hyperlipemia.
Restenosis following transluminal angioplasty in experimental atherosclerosis.
Authors: Faxon D P , Sanborn T A , Weber V J , Haudenschild C , Gottsman S B , McGovern W A , Ryan T J
Published: June 2011

Percutaneous transluminal angioplasty has received considerable attention in the treatment of obstructive atherosclerotic lesions in humans. However, restenosis frequently occurs and has limited the long-term effectiveness of this procedure. To study restenosis, a model of atherosclerosis was developed in 16 New Zealand rabbits. Atherosclerosis was induced in one or both iliac vessels by balloon deendothelialization followed by a 2% cholesterol diet for 6 weeks. Angiographic lesions were demonstrable in all animals. Fourteen iliac vessels served as controls, and nine underwent successful angioplasty with an increase in luminal diameter from 1.0 +/- 0.2 to 1.9 +/- 0.4 mm (p less than 0.01). After 4 weeks on a high cholesterol diet, all animals had another angiogram, which documented significant progression of disease in only six of 14 control iliac vessels, but in all nine dilated vessels. The average decrease in luminal diameter was 0.2 +/- 0.3 mm for the control group compared with 1.6 +/- 0.5 mm for the dilated group (p less than 0.01). Histopathological correlates revealed further remodeling of at various stages of organization and recanalization. In summary, this study demonstrates that restenosis occurs following transluminal angioplasty and is significantly more frequent than the natural progression of disease in a rabbit model of atherosclerosis. The mechanism of this restenosis appears to be related to intraluminal thrombosis and acceleration of atherosclerosis. Evaluation of antiplatelet drugs in the prevention of restenosis seems warranted.
Absence of lipoprotein lipase in cultured human adipose stromal cells.
Authors: Kern P A , Eckel R H
Published: June 2011

To develop a system for studying the regulation of adipose tissue lipoprotein lipase (LPL) in human cells, we isolated cells from the stromalvascular fraction of human adipose tissue and propagated them in tissue culture. These cells were cultured in medium containing fetal bovine, horse, or human sera in the absence or presence of aminoglycoside antibiotics. After the cells had been at confluence for at least 1 week, they were exposed to medium containing insulin, 3-isobutyl-l-methylxanthine (IBMX), and dexamethasone, in an attempt to induce differentiation into cells which produce LPL. Stromal cells in 62 cultures from 20 subjects were monitored for any LPL activity secreted into the culture medium. In addition, cultures were assayed for activity releasable with heparin, and extractable from cell digests in nonionic detergent. Upon reaching confluence, the human adipose tissue stromal cells began to accumulate lipid droplets. However, no consistent LPL activity was measured in the culture medium or after exposure to heparin. Five cultures contained a minimal amount of detergent extractable hydrolytic activity. In contrast, cultures of rat stromal cells not exposed to insulin, IBMX, and dexamethasone, contained assayable LPL in the culture medium (3.3 +/- 1.6 nEq/min/ml) and in the heparin-releasable fraction (1.7 +/- 0.5 nEq/min/10(6)cells). Thus, human adipose tissue stromal cells do not represent a useful system for studying the regulation of LPL. Although these cells accumulate some lipid, the absence of measurable LPL suggests that complete differentiation has not yet occurred.