bluuebunny/crossref_metadata_2025 huggingface dataset:
Smooth Muscle Cells Express Granulocyte-Macrophage Colony-Stimulating Factor in the Undiseased and Atherosclerotic Human Coronary Artery
Authors: Plenz Gabriele , Koenig Carsten , Severs Nicholas J. , Robenek Horst
Published: June 2011

AbstractGranulocyte-macrophage colony-stimulating factor (GM-CSF), one of a family of cytokines that regulate proliferation in macrophages and other types of cells, has been implicated in the inflammatory-fibroproliferative response of atherosclerosis. However, previous studies have been restricted to cultured cells and animal models. In the present study, we investigated GM-CSF expression in undiseased and atherosclerotic human coronary arteries at both the mRNA and protein levels. Dual in situ hybridization/cell-marking experiments demonstrated that subpopulations of intimal smooth muscle cells (SMCs) and endothelial cells express the cytokine in the histologically normal human coronary artery and that augmented expression occurs at these sites, and in macrophage accumulations and medial SMCs, in the atherosclerotic vessel. Corresponding data were obtained by in situ hybridization and reverse transcription–polymerase chain reaction and Northern analyses of cultured cells. Cultured human coronary arterial SMCs showed constitutive expression of GM-CSF in cells that had adopted an activated synthetic phenotype. Electron microscope immunocytochemistry revealed that GM-CSF is a protein localized in the cytoplasmic matrix of SMCs of both the undiseased and atherosclerotic vessel wall; extracellular matrix was largely unlabeled, with only occasional small patches of amorphous immunopositive material. The expression of GM-CSF by subpopulations of intimal SMCs in the undiseased artery and the marked upregulation of GM-CSF apparent in atherosclerotic lesions suggest roles for the cytokine in the cellular events underlying initiation and progression of the human atherosclerotic lesion.
Interaction of Very-Low-Density, Intermediate-Density, and Low-Density Lipoproteins With Human Arterial Wall Proteoglycans
Authors: Anber V. , Millar J. S. , McConnell M. , Shepherd J. , Packard C. J.
Published: June 2011

Abstract
The specific interaction of lipoproteins with arterial wall constituents, particularly proteoglycans (APG), is believed to play an important role in the development of atherosclerosis. The objective of this study was to examine the interaction of apolipoprotein B (apoB) containing lipoprotein subfractions (VLDL1,
S
f
60 to 400; VLDL2,
S
f
20 to 60; IDL1,
S
f
16 to 20; IDL2,
S
f
12 to 16; LDLA,
S
f
8 to 12; and LDLB,
S
f
0 to 8) prepared by cumulative density gradient centrifugation with chondroitin sulfate-rich APG. Eighteen subjects were studied, and a similar pattern of interaction between the lipoprotein species and APG was found in all. The order of reactivity (as measured by increased turbidity due to insoluble complex formation) was IDL
S
f
12 to 16 ≥ LDL
S
f
8 to 12 > LDL
S
f
0 to 8 > IDL S
f
16 to 20 >> VLDL
S
f
20 to 60 > VLDL
S
f
60 to 400. When the subjects were divided on the basis of their LDL subfraction profile, the extent of insoluble complex formation was highest in the group in which small, dense LDLIII was predominant; intermediate in the group whose LDL was mainly LDLII; and lowest in the group with a high proportion of LDLI (the mean reactivity, AU at 600 nm, of APG with IDL
S
f
12 to 16 and LDL
S
f
8 to 12 was 0.66; 0.62 and 0.46, 0.43 and 0.20, and 0.21 for the three groups, respectively). Fibrate lipid-lowering treatment decreased the percentage of LDLIII and increased the percentage of LDLI within total LDL and reduced the reactivity of all apoB-containing lipoprotein fractions toward APG. Sialic acid content varied in different lipoprotein subfractions, being the highest in VLDL and lowest in LDL. However, across lipoprotein species, it did not significantly correlate with APG-binding reactivity, suggesting that other factors are important in determining the interaction of lipoproteins with APG. Modification of LDL arginine and lysine residues abolished the ability of the lipoprotein to interact with APG, a finding that supports the hypothesis that the interaction is dependent on key positively charged amino acids on apoB. These findings demonstrate that (1) the overall reactivity of apoB-containing lipoproteins is greatest in individuals with small, dense LDL and (2) within an individual, IDL of
S
f
12 to 16 is the most reactive species, and this may in part explain the positive correlation between IDL and risk of coronary heart disease.
Dose-Dependent Suppression of Transplant Arteriosclerosis in Aorta-Allografted, Cholesterol-Clamped Rabbits
Authors: Andersen Henrik Ørbaek , Holm Pernille , Stender Steen , Hansen Birgit Fischer , Nordestgaard Børge Grønne
Published: June 2011

Abstract
Cyclosporine may suppress transplant arteriosclerosis; however, it also raises plasma cholesterol, which could promote the disease. Our aim was to test these hypotheses experimentally. In experiment 1 (n=34), cholesterol was clamped at a human level of 5 to 7 mmol/L, and rabbits were given either saline or cyclosporine in a low, medium, or high dose. In experiment 2 (n=15), in which dietary cholesterol was fixed at 0.05 g·kg
−1
·d
−1
, and experiment 3 (n=16), in which no dietary cholesterol was added to the chow, rabbits were given either medium-dose cyclosporine, saline, or vehicle. The duration of each experiment was 5 weeks. In experiment 1, cyclosporine attenuated the development of transplant arteriosclerosis dose dependently (trend test:
P
<.0001). Cyclosporine also suppressed, in a dose-dependent manner, the activation of the immune system (trend test:
P
<.05) and the presence of T lymphocytes (trend test:
P
<.0001) and macrophages in the intima (trend test:
P
<.01). Despite a higher plasma cholesterol level in cyclosporine-treated rabbits compared with saline-treated rabbits in both experiment 2 (4.9 versus 2.9 mmol/L) and experiment 3 (1.6 versus 0.8 mmol/L), transplant arteriosclerosis was significantly reduced by cyclosporine (Mann-Whitney
U
test:
P
<.05 and
P
<.05). These results suggest that cyclosporine suppresses experimental transplant arteriosclerosis dose dependently. Accordingly, in the assessment of the optimal cyclosporine dose to heart-transplanted patients, it should be taken into account that a dose reduction may promote transplant arteriosclerosis.
Elevated Fasting Total Plasma Homocysteine Levels and Cardiovascular Disease Outcomes in Maintenance Dialysis Patients
Authors: Bostom Andrew G. , Shemin Douglas , Verhoef Petra , Nadeau Marie R. , Jacques Paul F. , Selhub Jacob , Dworkin Lance , Rosenberg Irwin H.
Published: June 2011

Abstract
There is an excess prevalence of hyperhomocysteinemia in dialysis-dependent end-stage renal disease (ESRD) patients. Cross-sectional studies of the relationship between elevated total homocysteine (tHcy) levels and prevalent cardiovascular disease (CVD) in this patient population suffer from severe methodologic limitations. No prospective investigations examining the association between tHcy levels and the subsequent development of arteriosclerotic CVD outcomes among maintenance dialysis patients have been reported. To assess whether elevated plasma tHcy is an independent risk factor for incident CVD in dialysis-dependent ESRD patients, we studied 73 maintenance peritoneal dialysis or hemodialysis patients who received a baseline examination between March and December 1994, with follow-up through April 1, 1996. We determined the incidence of nonfatal and fatal CVD events, which included all validated coronary heart disease, cerebrovascular disease, and abdominal aortic/lower-extremity arterial disease outcomes. After a median follow-up of 17.0 months, 16 individuals experienced at least one arteriosclerotic CVD event. Cox proportional-hazards regression analyses, unadjusted and individually adjusted for creatinine, albumin, and total cholesterol levels, total/HDL cholesterol ratio, dialysis adequacy/residual renal function, baseline CVD, and the established CVD risk factors (ie, age, sex, smoking, hypertension, diabetes/glucose intolerance, and dyslipidemia) revealed that tHcy levels in the upper quartile (≥27.0 μmol/L) versus the lower three quartiles (<27.0 μmol/L) were associated with relative risk estimates (hazards ratios, with 95% confidence intervals for the occurrence of (pooled) nonfatal and fatal CVD ranging from 3.0 to 4.4; 95% confidence intervals (1.1-8.1) to (1.6-12.2). We conclude that the markedly elevated fasting tHcy levels found in dialysis-dependent ESRD patients may contribute independently to their excess incidence of fatal and nonfatal CVD outcomes.
Role of Lipoprotein-Bound NEFAs in Enhancing the Specific Activity of Plasma CETP in the Nephrotic Syndrome
Authors: Braschi Sylvie , Masson David , Rostoker Guy , Florentin Emmanuel , Athias Anne , Martin Claude , Jacotot Bernard , Gambert Philippe , Lallemant Christian , Lagrost Laurent
Published: June 2011

Abstract
Plasma cholesteryl ester transfer protein (CETP) activity, evaluated by the transfer of radiolabeled cholesteryl esters from a tracer dose of tritiated HDL to the plasma apolipoprotein B–containing lipoproteins, was significantly higher in patients with untreated idiopathic nephrotic syndrome (n=15) than in normolipidemic control subjects (n=22) (81.5±8.4 versus 43.1±3.1 μg CE · mL
−1
· h
−1
, respectively;
P
<.001). The increased CETP activity in nephrotic plasma was explained by a significant rise in both the CETP mass concentration (3.2±0.2 versus 2.1±0.1 mg/L;
P
<.001), and the specific CETP activity, calculated as the ratio of CETP activity to CETP mass (25.3±1.7 versus 20.4±1.6 μg CE · mg
−1
· h
−1
;
P
<.05). Elevated CETP activity in nephrotic patients was shown to be associated with a significant decrease in the mean size of LDL (24.4±0.5 versus 26.3±0.5 nm;
P
<.0001) as well as in the relative abundance of HDL
2a
(29.6±1.6% versus 34.8±1.1%;
P
<.05). The nephrotic syndrome was characterized by a significant increase in the relative proportion of lipoprotein-bound nonesterified fatty acids (NEFAs) (35.4±7.7% versus 7.6±3.0% of total;
P
<.01), leading to a significant increase in the electronegative charge of LDL (−4.3±0.1 versus −3.9±0.1 mV;
P
<.05) and HDL (−11.5±0.1 versus −11.1±0.2 mV;
P
<.05). Compared with native, nonsupplemented plasma, removal of lipoprotein-bound NEFAs by addition of fatty acid–poor albumin to total plasma from nephrotic patients or control subjects significantly decreased CETP activity and specific CETP activity. Specific CETP activity no longer differed between nephrotic and control groups after albumin supplementation (19.7±1.5 versus 17.7±1.5 μg CE · mg
−1
· h
−1
; NS). It is concluded that, in addition to elevated CETP mass concentration, lipoprotein-bound NEFAs, by increasing the negative electrostatic charge of nephrotic lipoproteins, can facilitate the CETP-mediated neutral-lipid transfer reaction in total plasma from nephrotic patients.
Regulation of Tumor Necrosis Factor-α and Interleukin-1β Induced Adhesion Molecule Expression in Human Vascular Smooth Muscle Cells by cAMP
Authors: Braun Marina , Pietsch Peter , Zepp Angela , Schrör Karsten , Baumann Gert , Felix Stephan B.
Published: June 2011

AbstractThis study investigates the hypothesis that the elevation of intracellular cAMP may affect cytokine-induced expression of adhesion molecules on human vascular smooth muscle cells. In cultured human smooth muscle cells from coronary arteries and saphenous veins, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induced the expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), whereas interferon-γ (INF-γ) selectively stimulated the expression of ICAM-1. Adenylyl cyclase was stimulated either by the stable prostacyclin mimetic cicaprost or by forskolin. Adhesion molecules were detected by a cell surface enzyme immunoassay and the respective mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Cicaprost as well as forskolin significantly inhibited TNF-α- and IL-1β-induced cell surface expression of ICAM-1 and VCAM-1. Semiquantitative rt-PCR measurements showed a marked decrease of TNF-α- and IL-1β-induced mRNA levels of both adhesion molecules after preincubation with cicaprost. The stability of TNF-α-induced ICAM-1 and VCAM-1 expression at mRNA and protein level was not altered by cicaprost. The IFN-γ-induced increase of cell surface expression of ICAM-1 and the respective mRNA levels, however, were not significantly altered by elevation of intracellular cAMP. Basal and stimulated cAMP levels, measured by radioimmunoassay, did not differ in TNF-α- and IFNγ-treated cells. The present results demonstrate that the expression of adhesion molecules on human smooth muscle cells induced by cytokines is differentially modulated by activation of adenylyl cyclase.
Moderate Genetic Influences on Plasma Levels of Plasminogen Activator Inhibitor-1 and Evidence of Genetic and Environmental Influences Shared by Plasminogen Activator Inhibitor-1, Triglycerides, and Body Mass Index
Authors: Hong Yuling , Pedersen Nancy L. , Egberg Nils , de Faire Ulf
Published: June 2011

Abstract
Both genes and environmental factors have been reported to influence plasma levels of plasminogen activator inhibitor-1 (PAI-1). However, the relative importance of genetic influences (ie, heritability) on plasma PAI-1 levels has not yet been investigated. Furthermore, PAI-1 levels are correlated with body mass index (BMI) and triglycerides. These correlations could reflect genetic and/or environmental factors in common to PAI-1, triglycerides, and BMI. We applied multivariate genetic analysis methods to assess the relative importance of genetic and environmental influences on plasma PAI-1 levels and to test the significance of genetic and/or environmental influences shared by PAI-1, triglycerides, and BMI in 217 pairs of middle-aged and elderly twins, of whom 113 pairs were reared apart and 121 pairs were women. The heritability estimate for PAI-1 levels was 42%. Individual-specific environmental factors explained 36% of the variance for PAI-1 levels. The remaining variance of PAI-1 was explained by rearing and residual-familial environmental factors. Furthermore, a genetic correlation of 1.00 between PAI-1 and triglycerides, a rearing environmental correlation of 1.00 between PAI-1 and BMI, a residual-familial environmental correlation of 1.00 between PAI-1 and triglycerides, and a genetic correlation of 0.63 between PAI-1 and BMI, were found. In conclusion, the present results suggest that genetic influences on plasma PAI-1 are moderate. Genetic and shared rearing or residual-familial environmental factors shared by PAI-1, BMI, and triglycerides explain the phenotypic association between these measures. It appears that all the genetic influences for PAI-1 are more or less shared with those for triglycerides and BMI.
Long-term Probucol Treatment Reverses the Severity of Myocardial Injury in Watanabe Heritable Hyperlipidemic Rabbits
Authors: Hoshida Shiro , Yamashita Nobushige , Igarashi Junsuke , Aoki Kazuhiro , Kuzuya Tsunehiko , Hori Masatsugu
Published: June 2011

Abstract
We previously reported that administration of NO donors ameliorates the severity of myocardial injury in cholesterol-fed rabbits. We now evaluated the effects of probucol, a lipid-lowering antioxidant that can preserve endothelium-dependent relaxation (EDR), in the aortas of cholesterol-fed rabbits. We examined the effects of short-term (7 days) or long-term (24 weeks) administration of 1% probucol on the size of infarcts resulting from 30 minutes of coronary occlusion followed by reperfusion (for 48 hours) in Watanabe heritable hyperlipidemic (WHHL) rabbits. Infarcts in untreated WHHL rabbits were significantly larger than those in the rabbits receiving the long-term but not the short-term treatment with probucol (72.2±5.4%, 37.6±6.4%, and 66.7±3.5%, respectively). Long-term probucol treatment also significantly reduced myeloperoxidase activity in both ischemic and nonischemic myocardium and suppressed P-selectin expression in the coronary vasculature. No significant differences were observed in hemodynamic parameters during myocardial ischemia/reperfusion. Long-term probucol treatment significantly reduced the surface area of atherosclerotic plaque lesions in the aorta (24.4±3.8% vs 46.3±6.3,
P
<.05). Moreover, long-term probucol treatment restored acetylcholine-induced EDR in aortic rings but did not affect sodium nitroprusside–induced relaxation. Finally, long-term probucol treatment resulted in significantly elevated cGMP levels in the aorta. These results indicate that long-term probucol treatment significantly ameliorates myocardial injury in heritable atherosclerotic rabbits, perhaps by reducing the accumulation of leukocytes in the myocardium and atherosclerotic vascular lesions. Thus, long-term administration appears to suppress the progression of atherosclerotic vascular disease in this animal model.
The Apolipoprotein E and β-Fibrinogen G/A-455 Gene Polymorphisms Are Associated With Ischemic Stroke Involving Large-Vessel Disease
Authors: Kessler Christof , Spitzer Carsten , Stauske Dorothea , Mende Sabine , Stadlmüller Jörg , Walther Reinhard , Rettig Rainer
Published: June 2011

AbstractThe relationship between the apolipoprotein E (apoE) and β-fibrinogen G/A-455 polymorphisms and cerebrovascular disease (CVD) was examined in the present study. We compared 227 patients with the subtypes of CVD (large-vessel disease, lacunar stroke, cardiac embolism, or undetermined pathomechanisms) with 225 control subjects. The occurrence of apoE isoforms (E2, E3, and E4) and the β-fibrinogen G/A-455 genotype was determined in these individuals. No differences in apoE polymorphisms or allele frequencies between the CVD patients and control subjects were found. However, analysis of apoE genotypes as a function of stroke subtype revealed that the apoE4 allele was significantly more common in those patients with macroangiopathy-associated CVD. The only CVD risk factor that distinguished patients with the E4 allele from those with other apoE genotypes was elevated cholesterol. No association between the β-fibrinogen G/A-455 polymorphism and CVD was found. However, homozygosity for the A allele was more common in patients with CVD resulting from large-vessel disease. These data demonstrate that the apoE4 allele and the AA genotype of the β-fibrinogen G/A-455 polymorphism occur significantly more frequently in patients with CVD resulting from stenosis of large, brain-supplying vessels. Such genetic analyses may further our understanding of the etiology of cerebrovascular disease.
Effect of Advanced Glycation End Product–Modified Albumin on Tissue Factor Expression by Monocytes
Authors: Khechai F. , Ollivier V. , Bridey F. , Amar M. , Hakim J. , de Prost D.
Published: June 2011

Abstract
Diabetes is associated with a hypercoagulable state that contributes to macrovascular complications, including cardiovascular events. The glycation reaction, a consequence of chronic hyperglycemia, has also been implicated in the pathogenesis of diabetic complications. Glycated proteins have receptors on monocytes and generate reactive oxygen species that can regulate the expression of a number of genes. As abnormal monocyte expression of tissue factor (TF), the main initiator of the coagulation cascade, is responsible for thrombosis in a number of clinical settings, we studied the effect of glycated albumin on monocyte TF expression. Mononuclear cells were incubated with glycated albumin for 24 hours, and monocyte TF activity was measured with a plasma recalcification time assay; TF antigen was measured by ELISA and TF mRNA by RT-PCR. Glycated albumin induced blood monocyte expression of the procoagulant protein TF at the mRNA level. Oxidative stress appeared to be involved in this effect, as the antioxidant
N
-acetylcysteine diminished TF mRNA accumulation in stimulated monocytes. Hydroxyl radicals, which may be generated inside cells from H
2
O
2
via the Fenton reaction, also appeared to be involved in this effect, as hydroxyl radical scavengers downregulated TF activity and antigen levels (but not TF mRNA). Finally, the involvement of activated protein tyrosine kinase in the transmission of the signal from the membrane to the nucleus was suggested by the inhibitory effect of herbimycin A. These results point to a new mechanism for the hypercoagulability often described in diabetic patients and suggest that antioxidants or protein tyrosine kinase inhibitors might be of therapeutic value in this setting.
Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA
Authors: Luchoomun Jayraz , Zhou Zhangyin , Bakillah Ahmed , Jamil Haris , Hussain M. Mahmood
Published: June 2011

Abstract
Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (>50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (>80%) by triacsin C due to >60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.
Tissue Factor Pathway Inhibitor in Endothelial Cells Colocalizes With Glycolipid Microdomains/Caveolae
Authors: Lupu Cristina , Goodwin Christopher A. , Westmuckett Andrew D. , Emeis Jeff J. , Scully Michael F. , Kakkar Vijay V. , Lupu Florea
Published: June 2011

Abstract
Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor•factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-
3
H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol–phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface–associated inhibitory activity toward tissue factor•factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.
Conformational Changes in Apolipoprotein B Modulate Intracellular Assembly and Degradation of ApoB-Containing Lipoprotein Particles in HepG2 Cells
Authors: Macri Khosrow Adeli Joseph
Published: June 2011

Abstract
The linkage between the conformation of apolipoprotein B
100
(apoB) and the intracellular assembly and degradation of apoB-containing lipoproteins was investigated in the present study. Disruption of disulfide bond formation in newly synthesized apoB molecules through the use of the reducing agent DTT resulted in a decrease in the secretion of apoB-containing lipoproteins from HepG2 cells compared with control cells. The synthesis of total apoB (apoB
100
plus nascent chains), as well as a number of control proteins, such as albumin and α1-antitrypsin, was decreased significantly in DTT-treated cells. However, the intracellular accumulation of full-length apoB
100
molecules was not inhibited in the presence of DTT. Subcellular fractionation indicated that apoB molecules isolated from the microsomes of DTT-treated cells had an increased association with the microsomal membrane compared with apoB isolated from untreated cells. Analysis of the distribution of apoB-containing lipoproteins from the lumen of isolated microsomes demonstrated that in the presence of DTT, there was a shift in the distribution, such that there was a decrease in the formation of HDL-sized (lipid-poor) apoB-containing lipoproteins and a decrease in the formation of LDL/VLDL apoB particles. Alterations in apoB conformation and their impact on degradation were also investigated by using DTT and by inhibiting
N
-linked glycosylation with tunicamycin. DTT appeared to change the rate and pattern of apoB degradation. Degradation was accelerated in both intact and permeabilized HepG2 cells. ApoB degradation occurred in DTT-treated permeabilized cells without the usual generation of the 70-kD and 335-kD fragments and was largely
N
-acetyl-leucyl-leucyl-norleucinal (ALLN) insensitive. In tunicamycin-treated cells, DTT further accelerated the degradation of unglycosylated apoB. Overall, the data suggest that the misfolding of apoB may prevent the proper association of apoB with lipids, resulting in impairment of the assembly of mature apoB-containing lipoproteins. Alteration in the conformation of apoB also appears to alter the degradation pathway of apoB, such that the protein is degraded through a pathway that is at least in part ALLN insensitive.
Two Intracellular Signaling Pathways for Activation of Protein Kinase C Are Involved in Oxidized Low-Density Lipoprotein–Induced Macrophage Growth
Authors: Matsumura Takeshi , Sakai Masakazu , Kobori Shozo , Biwa Takeshi , Takemura Toru , Matsuda Hirofumi , Hakamata Hideki , Horiuchi Seikoh , Shichiri Motoaki
Published: June 2011

Abstract
Recent studies demonstrated that oxidized LDL (Ox-LDL) induces macrophage growth in vitro. The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth. Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC). Pertussis toxin completely inhibited the Ox-LDL–induced rise in free calcium ion and significantly inhibited macrophage growth by 50%. Moreover, PKC inhibitors calphostin C and H-7 significantly inhibited Ox-LDL–induced macrophage growth by 80%. On the other hand, phospholipase A2–treated acetylated LDL did not induce a rise in calcium but significantly activated PKC and led to significant macrophage growth that was significantly inhibited by calphostin C by 90%. These results suggest the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium that was mediated by pertussis toxin–sensitive G protein and the internalization of lysophosphatidylcholine through the scavenger receptors. These two pathways may play an important role in Ox-LDL–induced macrophage growth.
Apolipoprotein A-I
FIN
(Leu159→Arg) Mutation Affects Lecithin
Authors: Miettinen Helena E. , Jauhiainen Matti , Gylling Helena , Ehnholm Sonja , Palomäki Ari , Miettinen Tatu A. , Kontula Kimmo
Published: June 2011

Abstract
We showed earlier that the apolipoprotein A-I Leu159→Arg mutation (apoA-I
Fin
) results in dominantly inherited hypoalphalipoproteinemia. In the present study we investigated the effect of the apoA-I
Fin
mutation on lipoprotein profile, apoA-I kinetics, lecithin:cholesterol acyltransferase (LCAT) activation, and cholesterol efflux in vitro. Carriers (n=9) of the apoA-I
Fin
mutation exhibited several lipoprotein abnormalities. The serum HDL cholesterol level was diminished to 20% of normal, and nondenaturing gradient gel electrophoresis of HDL showed disappearance of particles at the 9.0- to 12-nm size range (HDL
2
-type) and the presence of small 7.8- to 8.9-nm (mostly HDL
3
-type) particles only. HDL
3
-type particles from both the mutation carriers and nonaffected family members were similarly converted to large, HDL
2
-type particles by phospholipid transfer protein in vitro. Studies on apoA-I kinetics in four affected subjects favored accelerated catabolism of apoA-I. Experiments with reconstituted proteoliposomes showed that the capacity of apoA-I
Fin
protein to activate LCAT was reduced to 40% of that of the wild-type apoA-I. The impact of the apoA-I
Fin
protein on cholesterol efflux was examined in vitro using [
3
H]cholesterol-loaded human fibroblasts and three different cholesterol acceptors: (1) total HDL, (2) total apoA-I combined with phospholipid, and (3) apoA-I isoform (apoA-I
Fin
or wild-type apoA-I isoform 1) combined with phospholipid. ApoA-I
Fin
did not impair phospholipid binding or cholesterol efflux from fibroblasts to any of the acceptors used. Only one of the nine apoA-I
Fin
carriers appears to have evidence of clinically manifested atherosclerosis. In conclusion, although the apoA-I
Fin
mutation does not alter the properties of apoA-I involved in promotion of cholesterol efflux, its ability to activate LCAT in vitro is defective. In vivo, apoA-I
Fin
was found to be associated with several lipoprotein composition rearrangements and increased catabolism of apoA-I.
Cholesterol-Mediated Changes of Neutral Cholesterol Esterase Activity in Macrophages
Authors: Miura Shinji , Chiba Tsuyoshi , Mochizuki Norihiro , Nagura Hiromi , Nemoto Kiyomitsu , Tomita Isao , Ikeda Masahiko , Tomita Takako
Published: June 2011

Abstract
Cholesteryl esters (CE) in lipid droplets undergo a continual cycle of hydrolysis and reesterification by neutral cholesterol esterase (N-CEase) and acyl CoA:cholesterol acyltransferase (ACAT), respectively. The mechanism by which HDL mobilizes CE from lipid droplets in J774 A.1 cells was investigated, focusing on N-CEase activity. We asked whether HDL enhances the activity and, if so, what signals induce the change of the activity. An incubation of cells with HDL enhanced the decline of cholesteryl-[1-
14
C]-oleate in foam cells and increased N-CEase activity in the supernatant of cell homogenate in a concentration-dependent manner, whereas incubation with LDL decreased the activity. In addition, N-CEase activity was fivefold higher when cells were cultured in 10% lipoprotein-deficient serum (LPDS) medium (2 μg cholesterol/mL) than when cultured in 10% fetal calf serum medium (31 μg cholesterol/mL), suggesting that changes in N-CEase activity are mediated by cholesterol. An addition of cholesterol (0 to 30 μg/mL) in LPDS medium markedly inhibited N-CEase activity with a concomitant increase in cellular cholesterol concentration. This inhibitory effect of cholesterol was also observed in mouse peritoneal macrophages. In vitro addition of cholesterol did not affect N-CEase activity. Treatment of cells with HMG-CoA reductase inhibitors enhanced N-CEase activity, whereas ACAT inhibitor decreased the activity. Northern blot analysis of N-CEase mRNA showed that the expression was not altered by the presence of cholesterol in LPDS medium. These results suggest that cholesterol downregulates N-CEase activity, probably through cholesterol-dependent appearance of some factors.
Antithrombotic Efficacy of Inactivated Active Site Recombinant Factor VIIa Is Shear Dependent in Human Blood
Authors: Ørvim Una , Barstad R. Marius , Örning Lars , Petersen Lizette B. , Ezban Mirella , Hedner Ulla , Sakariassen Kjell S.
Published: June 2011

Abstract
Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR- rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 s
−1
(veins) and 650 s
−1
(medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 s
−1
, the IC
50
(50% inhibition) was 30 nmol/L, whereas at 650 s
−1
, the IC
50
was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8×10,
5
2.6×10
5
, and 1.8×10
5
m
−1
s
−1
, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.
Cafestol, the Cholesterol-Raising Factor in Boiled Coffee, Suppresses Bile Acid Synthesis by Downregulation of Cholesterol 7α-Hydroxylase and Sterol 27-Hydroxylase in Rat Hepatocytes
Authors: Post Sabine M. , de Wit Elly C. M. , Princen. Hans M. G.
Published: June 2011

Abstract
Consumption of boiled coffee raises serum cholesterol levels in humans. The diterpenes cafestol and kahweol in boiled coffee have been found to be responsible for the increase. To investigate the biochemical background of this effect, we studied the effects of cafestol and a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) on bile acid synthesis and cholesterol 7α-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes.

Dose-dependent decreases of bile acid mass production and cholesterol 7α-hydroxylase and sterol 27-hydroxylase activity were found, showing a maximal reduction of −91%, −79%, and −49% respectively, at a concentration of 20 μg/mL cafestol. The decrease in 7α-hydroxylase and 27-hydroxylase activity paralleled well the suppression of the respective mRNAs, being −79% and −77%, and −49% and −46%, respectively, at 20 μg/mL cafestol. Run-on data showed a reduction in 7α-hydroxylase and 27-hydroxylase gene transcriptional activity after incubation with cafestol. The mixture of cafestol/kahweol/isokahweol was less potent in suppression of bile acid synthesis and cholesterol 7α-hydroxylase. Cafestol (20 μg/mL) had no effect on lithocholic acid 6β-hydroxylase mRNA, another enzyme involved in bile acid synthesis. LDL-receptor, HMG-CoA reductase, and HMG-CoA synthase mRNAs were significantly decreased by cafestol (−18%, −20%, and −43%, respectively).

We conclude that cafestol suppresses bile acid synthesis by downregulation of cholesterol 7α-hydroxylase and of, to a lesser extent, sterol 27-hydroxylase in cultured rat hepatocytes, whereas kahweol and isokahweol are less active. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.
Effects of Oral and Transdermal Estrogen/Progesterone Regimens on Blood Coagulation and Fibrinolysis in Postmenopausal Women
Authors: Scarabin Pierre-Yves , Alhenc-Gelas Martine , Plu-Bureau Geneviève , Taisne Pascale , Agher Rachid , Aiach Martine
Published: June 2011

Abstract
Postmenopausal hormone replacement therapy is associated with a reduction in the incidence of coronary heart disease. However, inconclusive results have been reported with respect to the risk of stroke, and recent studies consistently showed an increased risk of venous thromboembolism in postmenopausal women using oral estrogen. There are surprisingly few interventional studies to assess the true effects of estrogen-progestin regimens on blood coagulation and fibrinolysis, and the impact of the route of estrogen administration on hemostasis has not been well documented. Therefore, we investigated the effects of oral and transdermal estradiol/progesterone replacement therapy on hemostatic variables. Forty-five healthy postmenopausal women, aged 45 to 64 years, were assigned randomly to one of the three following groups: cyclic oral or transdermal estradiol, both combined with progesterone, or no hormonal treatment. Hemostatic variables were assayed at baseline and after a 6-month period. Pairwise differences in the mean change between the three groups were compared using nonparametric tests. Oral but not transdermal estradiol regimen significantly increased the mean value of prothrombin activation peptide (F1+2) and decreased mean antithrombin activity compared with no treatment. Differences in fragment F1+2 levels between active treatments were significant. The oral estrogen group was associated with a significant decrease in both mean tissue-type plasminogen (t-PA) concentration and plasminogen activator inhibitor (PAI-1) activity and a significant rise in global fibrinolytic capacity (GFC) compared with the two other groups. A transdermal estrogen regimen had no significant effect on PAI-1, t-PA, and GFC levels. There were no significant changes in mean values of fibrinogen, factor VII, von Willebrand factor, protein C, fibrin D-dimer, and plasminogen between and within the three groups. We conclude that oral estrogen/progesterone replacement therapy may result in coagulation activation and increased fibrinolytic potential, whereas opposed transdermal estrogen appears without any substantial effects on hemostasis. Whereas these results may account for an increased risk of venous thromboembolism in users of oral postmenopausal estrogen, they emphasize the potential importance of the route of estrogen administration in prescribing hormone replacement therapy to postmenopausal women, especially to those at high risk of thrombotic disease.
Induction and Role of NO Synthase in Hypotensive Hepatic Failure
Authors: Smith Russell E.A. , Robinson Nicholas M.K. , McPeake James R. , Baylis Sally A. , Charles Ian G. , Heaton Nigel D. , Moncada Salvador , Williams Roger , Martin John F.
Published: June 2011

Abstract
Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by
N
G
-monomethyl-
l
-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.
Comparison of the Genetic Defect with LDL-Receptor Activity in Cultured Cells from Patients With a Clinical Diagnosis of Heterozygous Familial Hypercholesterolemia
Authors: Sun Xi-Ming , Patel Dilip D. , Knight Brian L. , Soutar Anne K.
Published: June 2011

AbstractIn this study we have analyzed the genetic defect in 42 patients with a diagnosis of heterozygous familial hypercholesterolemia (FH) by Southern blotting, SSCP, and sequencing of PCR-amplified fragments of genomic DNA or sequencing of RT-PCR products from mRNA in cultured cells. The apoB Arg3500Gln mutation was identified in five patients. A molecular defect in the LDL-receptor gene was confirmed in 23 patients; 16 of these mutations have not been described before. No defect in the coding region, intron:exon junctions or proximal promoter of the LDL-receptor gene or in the region of the apoB gene coding for the LDL-receptor binding domain was found in the remaining 14 patients. LDL-receptor activity and protein content of cultured lymphoblasts from the patients was significantly lower in cells from patients with severe rather than mild LDL-receptor mutations. Cells from four patients with no detectable defect showed reduced LDL receptor activity compared with eight normal cell lines, whereas six others had reduced LDL-receptor activity but LDL-receptor protein content within the normal range. Cells from four patients appeared to have normal LDL-receptor function. Cells from two patients with a defined defect also had LDL-receptor activity within the normal range. The findings demonstrate the problems involved in the genetic diagnosis of FH in patients. .
Genetic Contribution of the Endothelial Constitutive Nitric Oxide Synthase Gene to Plasma Nitric Oxide Levels
Authors: Wang Xing L. , Mahaney Michael C. , Sim Ah Siew. , Wang Jun , Wang Jian , Blangero John , Almasy Laura , Badenhop Renee B. , Wilcken David E. L.
Published: June 2011

Abstract
Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NO
x
: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NO
x
levels is due to genes (χ
2
[1]
=16.04,
P
=.000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NO
x
levels, and measured genotype analysis showed that plasma NO
x
levels in those homozygous for the rare allele (64.9±7.8 μmol/L) were significantly higher (
P
=.000242) than those homozygous for the common allele (30.2±3.1 μmol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NO
x
levels (
P
=.0066). While many environmental factors have been shown to alter transiently plasma NO
x
levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NO
x
, ie basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.
cAMP Involvement in the Expression of MMP-2 and MT-MMP1 Metalloproteinases in Human Endothelial Cells
Authors: Peracchia Francesca , Tamburro Antonio , Prontera Cesaria , Mariani Barbara , Rotilio Domenico
Published: June 2011

AbstractMatrix metalloproteinases (MMPs) are a multigene family of enzymes secreted by a variety of cells, including human umbilical vein endothelial cells (HUVECs). Because metalloproteinases are potentially destructive agents, their production is tightly controlled at several levels. Rather little is known about the presence and regulation of MMPs in endothelial cells. In this study, we investigated the expression and regulation of MMP-2 and membrane type-matrix metalloproteinase (MT-MMP1), a membrane metalloproteinase strictly related to MMP-2 activation. Zymographic analysis of conditioned medium (CM) of HUVECs showed the presence of gelatinolytic activity mainly at 72 and 64 and 62 kD. The 64- and 62-kD bands, respectively, represent the intermediate and the completely active forms of MMP-2. When HUVECs were treated with forskolin (FK) (100 and 25 μmol/l), there was a decrease in the appearance of the 64 to 62 kDa doublet, suggesting an inhibition of the fully activated form of MMP-2. FK raises intracellular cAMP in HUVECs. The same data were obtained using dibutyryl-cAMP. Northern analysis revealed that the expression of MMP-2 increased slightly after treatment with FK, in contrast with gelatin zymography results. Taking into consideration the mechanism of activation of MMP-2, we tested the hypothesis that this compound could modulate MT-MMP1. As expected, FK was able to decrease MT-MMP1 expression. These data correlate with experiments using membranes of FK-treated HUVECs and incubated with control CM. Zymography revealed that when CM was incubated with membranes prepared from FK-treated HUVECs, there was a decrease in the appearance of the 64-kDa band, suggesting that the expression of MT-MMP1 was negatively modified. These results correlate with the MT-MMP1 protein level, negatively modified after FK treatment.
Shear Stress–Induced Detachment of Blood Platelets From Various Surfaces
Authors: Wu Ya-Ping , de Groot Philip G. , Sixma Jan J.
Published: June 2011

Abstract
Platelet accumulation is the result of platelet adhesion and detachment. This study describes platelet detachment from fibronectin, laminin, fibrinogen, von Willebrand Factor (vWF), endothelial cell matrix (ECM), and collagen type III. Platelets adhered after 5 minutes’ perfusion of anticoagulated whole blood at different shear rates were subjected to a brief flush of 1 minute with HEPES-buffered saline at varying shear stress. Platelets adhering to fibronectin and laminin were most easily detached. Fibrinogen and vWF had an intermediate position. Almost no detachment occurred from ECM and collagen type III. Dendritic platelets were removed more easily than spread platelets. When the shear rate at which adhesion had occurred was raised, platelet detachment decreased strongly. When the time period between adhesion and detachment was increased, platelet detachment also decreased. From these results, we conclude that detachment is determined initially by the shear rate at which platelets adhere, then by the time they are allowed to settle, then by the nature of the surface, and then by the degree of spreading. The shear optimum for a given adhesive protein is not determined by the detachment.