bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Free Fatty Acid Suppression During Hyperinsulinemia Is a Characteristic Finding in Familial Combined Hyperlipidemia, but Insulin Resistance Is Observed Only in Hypertriglyceridemic Patients
Authors: Pihlajamäki Jussi , Karjalainen Leena , Karhapää Pauli , Vauhkonen Ilkka , Laakso Markku
Published: June 2011

Abstract
—Insulin resistance has been associated with hypertriglyceridemia, combined hyperlipidemia, and familial combined hyperlipidemia (FCHL). Whether all FCHL patients with different types of dyslipidemia have low insulin sensitivity has not been evaluated. We measured insulin sensitivity by the hyperinsulinemic euglycemic clamp with indirect calorimetry in 110 healthy controls and in 105 nondiabetic, FCHL family members: in 50 without dyslipidemia, in 19 with hypercholesterolemia (total cholesterol ≥7.7 mmol/L), in 22 with hypertriglyceridemia (total triglycerides ≥2.4 mmol/L in men 2.4 mmol/L in women), and in 14 with combined hyperlipidemia. During the hyperinsulinemic clamp, FCHL family members had higher free fatty acid levels than did controls (0.06±0.06 [mean±SD] in controls versus 0.16±0.11 in relatives without dyslipidemia versus 0.15±0.07 in hypercholesterolemic patients versus 0.29±0.14 in hypertriglyceridemic patients versus 0.27±0.17 mmol/L in patients with combined hyperlipidemia;
P
<0.001 after adjustment for age, sex, and body mass index). Relatives without dyslipidemia (16.4±4.4 μmol · kg
−1
· min
−1
,
P
=0.001) and patients with hypertriglyceridemia (12.8±3.8 μmol · kg
−1
· min
−1
,
P
<0.001) and with combined hyperlipidemia (13.7±3.1 μmol · kg
−1
· min
−1
,
P
<0.001) had lower rates of insulin-stimulated glucose oxidation than did controls (19.4±4.7 μmol · kg
−1
· min
−1
). Also, the rates of nonoxidative glucose disposal were lower in patients with hypertriglyceridemia (
P
=0.001) and combined hyperlipidemia (
P
=0.011) than in controls. In contrast, subjects with hypercholesterolemia and control subjects had similar rates of insulin-stimulated glucose uptake. We conclude that a defect in free fatty acid suppression during hyperinsulinemia, probably located in adipose tissue, is characteristic for all FCHL patients with varying types of dyslipidemia, whereas insulin resistance in skeletal muscle is observed only in FCHL patients with elevated triglyceride levels.
Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide–Dependent Mechanism
Authors: Chang Yong S. , Yaccino Jean Ann , Lakshminarayanan Sunitha , Frangos John A. , Tarbell John M.
Published: June 2011

Abstract
—This study addresses the role of nitric oxide (NO) and its downstream mechanism in mediating the shear-induced increase in hydraulic conductivity (L
p
) of bovine aortic endothelial cell monolayers grown on porous polycarbonate filters. Direct exposure of endothelial monolayers to 20-dyne/cm
2
shear stress induced a 4.70±0.20-fold increase in L
p
at the end of 3 hours. Shear stress (20 dyne/cm
2
) also elicited a multiphasic NO production pattern in which a rapid initial production was followed by a less rapid, sustained production. In the absence of shear stress, an exogenous NO donor,
S
-nitroso-
N
-acetylpenicillamine, increased endothelial L
p
2.23±0.14-fold (100 μmol/L) and 4.8±0.66-fold (500 μmol/L) at the end of 3 hours. In separate experiments, bovine aortic endothelial cells exposed to NO synthase inhibitors,
N
G
-monomethyl-
l
-arginine and
N
G
-nitro-
l
-arginine methyl ester, exhibited significant attenuation of shear-induced increase in L
p
in a dose-dependent manner. Inhibition of guanylate cyclase (GC) with LY-83,583 (1 μmol/L) or protein kinase G (PKG) with KT5823 (1 μmol/L) failed to attenuate the shear-induced increase in L
p
. Furthermore, direct addition of a stable cGMP analogue, 8-bromo-cGMP, had no effect in altering baseline L
p
, indicating that the GC/cGMP/PKG pathway is not involved in shear stress–NO–L
p
response. Incubation with iodoacetate (IAA), a putative inhibitor of glycolysis, dose-dependently increased L
p
. Addition of IAA at levels that did not affect baseline L
p
greatly potentiated the response of L
p
to 20-dyne/cm
2
shear stress. Finally, both shear stress–induced and IAA-induced increases in L
p
could be reversed with the addition of dibutyryl cAMP. However, additional metabolic inhibitors, 2 deoxyglucose (10 mmol/L) and oligomycin (1 μmol/L), or reactive oxygen species scavengers, deferoxamine (1 mmol/L) and ascorbate (10 mmol/L), failed to alter shear-induced increases in L
p
. Our results show that neither the NO/cGMP/PKG pathway nor a metabolic pathway mediates the shear stress–L
p
response. An alternate mechanism downstream from NO that is sensitive to IAA must mediate this response.
Retrovirus-Mediated, Stable Scavenger-Receptor Gene Transfer Leads to Functional Endocytotic Receptor Expression, Foam Cell Formation, and Increased Susceptibility to Apoptosis in Rabbit Aortic Smooth Muscle Cells
Authors: Lehtolainen Pauliina , Takeya Motohiro , Ylä-Herttuala Seppo
Published: June 2011

Abstract
—The type II, class A macrophage scavenger receptor (SR-A) plays an important role in the pathogenesis of atherosclerosis and foam cell formation. However, its role in nonmacrophage cell lines remains unknown. To test the hypothesis that SR-A activity leads to proatherogenic changes in nonmacrophage cell lines, we generated Moloney murine leukemia virus– and vesicular stomatitis virus G protein–pseudotyped retroviruses containing SR-A type II cDNA, which were used for stable transfection of SR-A activity into mouse fibroblasts and rabbit aortic smooth muscle cells (SMCs). β-Galactosidase–transfected cell lines were used as controls. Transfected cell lines expressed functional SR-A mRNA and protein. Expression of SR-A activity was stable for at least 9 months. By electron microscopy, transfected receptors were located in coated pits and in intracellular structures resembling endocytotic vesicles. Expression of SR-A on the cell surface was verified by flow cytometry and by uptake and degradation of
125
I-labeled acetylated low density lipoprotein (LDL). Increases of 5- to 25-fold and of 6- to 8-fold in the rate of acetylated LDL degradation were observed in transfected fibroblasts and SMCs, respectively, compared with β-galactosidase–transfected control cell lines. Incubation of the transfected SMCs and fibroblasts with acetylated or oxidized LDL led to foam cell formation. Incubation with oxidized LDL also led to increased apoptosis and cell death. An altered morphology with increased cell size and granularity was observed in the most active SR-A SMC clones. It is concluded that stable overexpression of SR-A leads to foam cell formation and other proatherogenic changes in nonmacrophage cell lines. Stable SMC and fibroblast cell lines can be used as models for foam cell formation. The results also suggest that increased SR activity may play an important role in SMC-related pathology in atherosclerotic arteries.
Platelet-Activating Factor Enhances Vascular Endothelial Growth Factor–Induced Endothelial Cell Motility and Neoangiogenesis in a Murine Matrigel Model
Authors: Montrucchio Giuseppe , Lupia Enrico , Battaglia Edda , Del Sorbo Lorenzo , Boccellino Mariarosaria , Biancone Luigi , Emanuelli Giorgio , Camussi Giovanni
Published: June 2011

Abstract—We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of125I-albumin. The synthesis of PAF, cell motility, and the increased125I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein–derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.
Anti-Human von Willebrand Factor Monoclonal Antibody AJvW-2 Prevents Thrombus Deposition and Neointima Formation After Balloon Injury in Guinea Pigs
Authors: Kageyama Shunsuke , Yamamoto Hiroshi , Yoshimoto Ryota
Published: June 2011

Abstract
—Immediately after angioplasty, platelet adhesion to the injured arterial wall and subsequent release of various mitogens may contribute to neointima formation. The purpose of this study was to evaluate the inhibitory effect of AJvW-2, a monoclonal antibody against human von Willebrand factor (vWF), on neointima formation in a guinea pig model. The carotid artery was injured with a balloon catheter, and AJvW-2 was administered by a single bolus injection. AJvW-2 dose-dependently prevented neointima formation 14 days after injury. Significant inhibition was observed at 1.8 mg/kg, at which dose significant inhibition of platelet aggregation was achieved for 2 days. By elastic-Masson staining, organized thrombi were observed in the neointimal lesion on day 14. The thrombus area was significantly correlated with neointimal thickness. Furthermore, thrombus deposition, immunostained for vWF and fibrin(ogen), was observed on the media immediately after balloon injury. AJvW-2 significantly reduced the deposition of both adhesive proteins and reduced the incidence of organized thrombus formation, which might affect subsequent neointima formation. However, the proliferation of cultured smooth muscle cells was not affected by AJvW-2. These results suggest that AJvW-2 prevents neointima formation by inhibition of initial platelet-mediated thrombus formation rather than by direct inhibition of smooth muscle cell proliferation.
Oxidation of Apolipoprotein B-100 in Circulating LDL Is Related to LDL Residence Time
Authors: Pietzsch Jens , Lattke Peter , Julius Ulrich
Published: June 2011

Abstract
—5-Hydroxy-2-aminovaleric acid (HAVA) has been suggested to be a specific marker of oxidation of apolipoprotein (apo) B-100 proline (Pro) and arginine (Arg) side-chain residues in low density lipoprotein (LDL) in vitro. Here we describe the application of sensitive mass spectrometric techniques to the characterization of Pro/Arg-modified apoB-100 in LDL
1
(S
f
7 to 12) and LDL
2
(S
f
0 to 7) in vivo. We studied 7 subjects with familial defective apoB-100 (FDB) and 8 normolipidemic controls. In FDB subjects, the presence of a mutant apoB-100 (FDB
3500Q
) in LDL markedly reduced its affinity for the LDL receptor, leading to increased residence times (RTs) of LDL
1
(65±21 versus 32±12 hours,
P
<0.005) and LDL
2
(230±40 versus 53±7 hours,
P
<0.001) when compared with controls, as determined by stable-isotope turnover studies. LDL
1
HAVA content was not different between the groups (FDB, 0.004±0.001 mol/mol apoB-100 versus controls, 0.003±0.001 mol/mol apoB-100,
P
=0.200). LDL
2
HAVA content was higher in FDB subjects (0.374±0.088 versus 0.013±0.002 mol/mol apoB-100,
P
<0.001). In both groups, LDL
2
HAVA was positively associated with LDL
2
RT (FDB,
r
=0.893,
P
=0.003; controls,
r
=0.976,
P
=0.000) and negatively correlated with LDL
2
α-tocopherol content (FDB,
r
=−0.929,
P
=0.003; controls,
r
=−0.903,
P
=0.002). No significant correlations could be found between LDL
1
HAVA, LDL
1
RT, and α-tocopherol, respectively. The low LDL
1
HAVA content observed in both FDB and control groups was thought to be due to the relatively lower RT as well as the higher α-tocopherol content of these lipoproteins. In contrast, LDL
2
seemed to be strongly prone to direct oxidation of apoB-100 in vivo. The longer these particles linger in the circulation, the more apoB-100 Pro/Arg residues become modified.
R3531C Mutation in the Apolipoprotein B Gene Is Not Sufficient to Cause Hypercholesterolemia
Authors: Rabès Jean-Pierre , Varret Mathilde , Devillers Martine , Aegerter Philippe , Villéger Ludovic , Krempf Michel , Junien Claudine , Catherine Boileau
Published: June 2011

Abstract—Familial hypercholesterolemia and familial ligand-defective apolipoprotein B-100 (FDB) are dominantly inherited disorders leading to impaired low-density lipoprotein receptor (LDLR) and apolipoprotein B-100 (APOB) interaction, plasma LDL elevation, and hypercholesterolemia. We previously identified the first French FDB-R3531C proband, a woman with very high total cholesterol, in a group of type IIa hypercholesterolemic families. We report here the investigation of her family at large that revealed the total absence of cosegregation with hypercholesterolemia. Six of the 10 subjects heterozygous for the R3531C mutation had plasma cholesterol lower than the 97.5th percentile for their age and gender, and mean cholesterol levels were not significantly different between affected and unaffected persons. Furthermore, 2 family members with similar high LDL-cholesterol levels were not carriers of the R3531C substitution, suggesting the implication of another mutation. Segregation analysis of the LDLR gene revealed statistically significant genetic linkage with hypercholesterolemia, and analysis of the proband LDLR gene led to the identification of the 664 proline to leucine defective mutation and its detection in all 6 hypercholesterolemic-related members of this family. Therefore, our results show that the family presents with familial hypercholesterolemia and give evidence that the R3531C substitution in the APOB gene is not an allelic variant leading to FDB. Furthermore, thorough analysis of our data suggests that the APOB-R3531C mutation enhances the hypercholesterolemic effect of the LDLR-P664L defect, suggesting that it is a susceptibility mutation.
Phosphatidylinositol 3-Kinase Signaling Is Important for Smooth Muscle Cell Replication After Arterial Injury
Authors: Shigematsu Kunihiro , Koyama Hiroyuki , Olson N. Eric , Cho Aesim , Reidy Michael A.
Published: June 2011

Abstract
—The phosphoinositide 3-kinase [PI(3)K] pathway is a key signaling pathway important for replication of mammalian cells. In this study, we examined the role of PI(3)K in smooth muscle cell (SMC) replication after balloon catheter injury of rat carotid arteries. Protein kinase B (PKB), a downstream target of PI(3)K, was phosphorylated at 30 and 60 minutes after injury and to a lesser degree after 6 hours and 1 and 2 days but not after 7 days. Wortmannin (10 μg per rat), a PI(3)K inhibitor, given to rats 60 and 5 minutes before and 11 hours after balloon injury, reduced the levels of phosphorylated PKB. SMC replication quantified between 24 to 48 hours was significantly reduced compared with control replication, as were the levels of cyclin D
1
. Wortmannin was also administered to rats between days 7 and 8 and between days 7 and 9 after balloon catheter injury. A reduction in levels of phosphorylated PKB was detected, but no decrease in the replication of intimal SMCs was observed in either experiment. These data demonstrate that the PI(3)K signal transduction pathway plays an important role in medial but not intimal SMC replication.
Methylenetetrahydrofolate Reductase 677
C
→
T
Mutation and Coronary Heart Disease Risk in UK Indian Asians
Authors: Chambers John C. , Ireland Helen , Thompson Elizabeth , Reilly Peter , Obeid Omar A. , Refsum Helga , Ueland Per , Lane David A. , Kooner Jaspal S.
Published: June 2011

Abstract
—Plasma homocysteine concentrations are elevated in UK Indian Asians and may contribute to twice as many coronary heart disease (CHD) deaths in this group compared with European whites. The mechanisms underlying elevated homocysteine concentrations among Indian Asians are not well understood. In this study, we have investigated the extent to which the methylenetetrahydrofolate reductase (MTHFR) 677
C
→
T
mutation accounts for elevated plasma homocysteine and increased CHD risk in Indian Asians compared with European whites. We investigated 454 male cases (with myocardial infarction or angiographically proven CHD: 224 Indian Asians, 230 European whites) and 805 healthy male controls (381 Indian Asians, 424 European whites). Fasting homocysteine concentrations, MTHFR 677
C
→
T
genotype, and conventional CHD risk factors were measured. The prevalence of homozygous MTHFR 677
T
in Indian Asian controls was less than one third that in European white controls (3.1% versus 9.7%,
P
<0.001). In Indian Asians, the
TT
MTHFR genotype was not associated with homocysteine concentrations and was not present in any of the Asian controls with hyperhomocysteinemia (>15 μmol/L). In contrast, among European whites, the
TT
MTHFR genotype was strongly related to elevated plasma homocysteine concentrations and was found in 27% of the European controls with hyperhomocysteinemia. Elevated homocysteine in Indian Asian compared with European white controls was accounted for by their reduced levels of B vitamins but not by the MTHFR 677
T
genotype. However, neither the
TT
MTHFR genotype nor B vitamin levels explained the elevated homocysteine concentrations in CHD cases compared with controls.
TT
MTHFR was not a risk factor for early-onset CHD in Indian Asians (odds ratio, 0.5; 95% confidence interval, 0.1 to 2.4;
P
=0.39), unlike in European whites (odds ratio, 2.1; 95% confidence interval, 1.1 to 4.1;
P
=0.02). We conclude that the MTHFR 677
T
mutation does not contribute to elevated plasma homocysteine concentrations or increased CHD risk in Indian Asians compared with European whites. Our results suggest that novel genetic defects and/or environmental factors influence homocysteine metabolism in Indian Asians residing in the United Kingdom.
Macrophage Cholesterol Metabolism, Apolipoprotein E, and Scavenger Receptor AI/II mRNA in Atherosclerosis-Susceptible and -Resistant Mice
Authors: Friedman Gideon , Ben-Yehuda Arie , Dabach Yedida , Hollander Gideon , Babaey Sharona , Ben-Naim Mazal , Stein Olga , Stein Yechezkiel
Published: June 2011

Abstract
—Female mice known to be susceptible (C57BL) and resistant (C3H and BALB/c) to diet-induced atherosclerosis were studied. Feeding of a cholate-containing atherogenic diet for 1 month resulted in an increase in plasma total cholesterol, little or no change in total phospholipids and high density lipoprotein (HDL) cholesterol, and a fall in HDL phospholipid, which was most pronounced in the C57BL strain. In elicited macrophages, cholesterol esterification was lower with acetylated low density lipoprotein (acLDL) and higher with β-very low density lipoprotein (β-VLDL) in C57BL than in C3H or BALB/C strains. In resident macrophages, acLDL enhanced cholesterol esterification more than did rabbit β-VLDL. With acLDL, more apolipoprotein E (apoE) was recovered in all macrophage cultures. In macrophages from chow-fed mice, most apoE was in the medium, whereas in mice fed an atherogenic diet, half of the apoE was in the cells. ApoE protein was highest in macrophages from BALB/c mice fed an atherogenic diet; an increase in apoE mRNA occurred in BALB/c and C3H macrophages. Scavenger receptor AI/II mRNA was significantly higher in macrophages from atherosclerosis-resistant mice. Thus, higher HDL phospholipid and plasma apoE levels (reported by others), together with high macrophage scavenger receptor AI/II mRNA, could inhibit accretion of cholesterol in the vessel wall in the 2 resistant strains.
Dietary Factor VII Activation Does Not Increase Plasma Concentrations of Prothrombin Fragment 1+2 in Patients With Stable Angina Pectoris and Coronary Atherosclerosis
Authors: Bladbjerg Else Marie , Münster Anna-Marie , Marckmann Peter , Keller Niels , Jespersen Jørgen
Published: June 2011

Abstract
—Studies in healthy subjects showed that blood coagulation factor VII (FVII) is activated postprandially after consumption of high-fat meals, but accompanying thrombin formation has not been demonstrated. In patients with coronary atherosclerosis, the arterial intima is supposed to present more tissue factor, the cofactor of FVII, to circulating blood; therefore, thrombin formation in response to FVII activation is more likely to occur in such patients. This hypothesis was tested in a randomized crossover study of 30 patients (aged 43 to 70 years) with stable angina pectoris and angiographically verified coronary atherosclerosis. They were served a low-fat (5% of energy from fat) breakfast and lunch and a high-fat (40% of energy from fat) breakfast and lunch on 2 different days. Venous blood samples were collected at 8:15
am
(fasting), 12:30
pm
, 2:00
pm
, 3:30
pm
, and 4:45
pm
and analyzed for triglycerides, activated FVII (FVIIa), FVII protein concentration (FVII:Ag), prothrombin fragment 1+2 (F1+2), and soluble fibrin. Triglyceride levels increased from fasting levels on both diets, but they increased most markedly on the high-fat diet. FVIIa and FVIIa/FVII:Ag increased with the high-fat diet and decreased with the low-fat diet. For both diets, FVII:Ag and F1+2 decreased slightly. No postprandial changes were observed for soluble fibrin. Postprandial mean values of triglycerides, FVIIa, FVII:Ag, and FVIIa/FVII:Ag were significantly higher for the high-fat diet than for the low-fat diet. Our findings confirm that high-fat meals cause immediate activation of FVII. The clinical implication is debatable because FVII activation was not accompanied by an increase in plasma F1+2 concentrations in patients with severe atherosclerosis. However, a local thrombin generation on the plaque surface cannot be excluded.
HIV Protease Inhibitors Stimulate Hepatic Triglyceride Synthesis
Authors: Lenhard James M. , Croom Dallas K. , Weiel James E. , Winegar Deborah A.
Published: June 2011

Abstract
—Hyperlipidemia may complicate the use of HIV protease inhibitors (PIs) in AIDS therapy. To determine the cause of hyperlipidemia, the effect of PIs on lipid metabolism was examined with HepG2 liver cells and AKR/J mice. In HepG2 cells, the PIs ABT-378, nelfinavir, ritonavir, and saquinavir stimulated triglyceride synthesis; ritonavir increased cholesterol synthesis; and amprenavir and indinavir had no effect. Moreover, nelfinavir increased mRNA expression of diacylglycerol acyltransferase and fatty acid synthase. The retinoid X receptor agonist LG100268, but not the antagonist LG100754, further increased PI-stimulated triglyceride synthesis and mRNA expression of fatty acid synthase in vitro. In fed mice, nelfinavir or ritonavir did not affect serum glucose and cholesterol, whereas triglyceride and fatty acids increased 57% to 108%. In fasted mice, ritonavir increased serum glucose by 29%, cholesterol by 40%, and triglyceride by 99%, whereas nelfinavir had no effect, suggesting these PIs have different effects on metabolism. Consistent with the in vitro results, nelfinavir and ritonavir increased triglyceride 2- to 3-fold in fasted mice injected with Triton WR-1339, an inhibitor of triglyceride clearance. We propose that PI-associated hyperlipidemia is due to increased hepatic triglyceride synthesis and suggest that retinoids or meal restriction influences the effects of select PIs on lipid metabolism.
ICAM-1 Deficiency Reduces Atherosclerotic Lesions in Double-Knockout Mice (ApoE
−
/−
/ICAM-1
−
/−
) Fed a Fat or a Chow Diet
Authors: Bourdillon Marie-Claude , Poston Robin N. , Covacho Chantal , Chignier Elza , Bricca Giampiero , McGregor John Louis
Published: June 2011

Abstract
—Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE
−
/−
mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE
−
/−
) or double-knockout (DKO; ApoE
−
/−
, ICAM-1
−
/−
) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.
Regulation of Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) Synthesis, Degradation, and Translocation by High-Density Lipoprotein
2
at a Low Concentration
Authors: Li Lei , Pownall Henry J.
Published: June 2011

Abstract
—Although plasma HDL
2
cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL
2
at a low concentration (≤40 μg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL
2
on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL
2
at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL
2
, however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL
2
promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL
2
is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.
Contribution of Apolipoprotein C-III Gene Variants to Determination of Triglyceride Levels and Interaction With Smoking in Middle-Aged Men
Authors: Waterworth Dawn M. , Talmud Philippa J. , Bujac Sarah R. , Fisher Rachel M. , Miller George J. , Humphries Steve E.
Published: June 2011

Abstract
—Variation within and around the apolipoprotein C-III (
APOC3
) gene has been associated with elevated triglyceride (Tg) levels and cardiovascular disease. The associations of 4 polymorphic variants in the
APOC3
gene (3238C>G in the 3′ untranslated region [
Sst
I], 1100C>T in exon 3, −482C>T in the insulin-responsive element, and −2854T>G in the
APOC3-A4
intergenic region) with plasma Tg and cholesterol levels and their interaction with smoking have been investigated in the Second Northwick Park Heart Study (NPHSII), a large cohort of healthy men (n=2745). Analyzing the variants separately showed that 3238G, 1100T, and −482T alleles were all associated with raised Tg levels. For the 3238C>G and −482C>T sites, the Tg-raising effect appeared to depend on smoking status (test for interaction,
P
=0.042 and
P
=0.009, respectively), but for the 1100C>T site, the effect was constant irrespective of smoking status (test for interaction,
P
=0.27). The −2854T>G site was not associated with effects on Tg levels in this sample. Because all of the variants showed significant allelic association, regression modeling was used to quantify the relative size of each effect and to assess whether the effects of the separate variants were independent. The 1100C>T variant had an independent effect on Tg levels that was not influenced by smoking status (increase of 8.2% in Tg with each T1100 allele), whereas the −482C>T variant had a separate effect that was dependent on smoking (increase of 13.7% in Tg for each −482T allele in current smokers, 8.6% in exsmokers, and −7.4% in those who never smoked). The 3238C>G variant did not show a separate independent effect on Tg concentration. Thus, by use of the regression model, it was possible to estimate how mean Tg levels would vary in groups of individuals with respect to
APOC3
genotype and smoking information. Analysis in this large group of healthy men has allowed the identification of a statistically robust
APOC3
genotype-smoking interaction, which now warrants further molecular study.
CD9 Participates in Endothelial Cell Migration During In Vitro Wound Repair
Authors: Klein-Soyer Claudine , Azorsa David O. , Cazenave Jean-Pierre , Lanza François
Published: June 2011

Abstract
—CD9, a widely expressed membrane protein of the tetraspanin family, has been implicated in diverse functions, such as signal transduction, cell adhesion, and cell motility. We tested the effects of an anti-CD9 monoclonal antibody (ALMA.1) on the migration and proliferation of human vascular endothelial cells (ECs) during repair of an in vitro mechanical wound mimicking angiogenic processes. ALMA.1 induced dose-dependent inhibition of wound repair with a 35±1.5% decrease at 20 μg/mL. Only cell migration was affected, because the rate of proliferation of ECs at the lesion margin was not modified and because the inhibition of repair was also observed for nonproliferating irradiated ECs. Monoclonal antibodies against CD63 tetraspanin (H5C6) and control mouse IgG (MOPC-21) were inactive. CD9, one of the most abundant proteins at the surface of ECs, colocalized with β
1
or β
3
integrins on EC membranes in double-labeling immunofluorescence experiments with ALMA.1 and an anti-β
1
(4B4) or anti-β
3
(SDF.3) monoclonal antibody. Moreover, ALMA.1 and 4B4 had additive inhibitory effects on lesion repair, whereas 4B4 alone also inhibited EC proliferation. In transmembrane Boyden-type assays, ALMA.1 induced dose-dependent inhibition of EC migration toward fibronectin and vitronectin with 45±6% and 31±10% inhibition, respectively, at 100 μg/mL. 4B4 inhibited migration toward fibronectin at 10 μg/mL but had no effect in the case of vitronectin. Adhesion of ECs to immobilized anti-CD9 monoclonal antibodies induced tyrosine-phosphorylated protein levels similar to those observed during interactions with β
1
or β
3
integrins. These results point to the involvement of CD9 in EC adhesion and migration during lesion repair and angiogenesis, probably through cooperation with integrins. As such, CD9 is a potential target to inhibit angiogenesis in metastatic and atherosclerotic processes.
Insulin-Mediated Stimulation of Protein Kinase Akt
Authors: Hermann Corinna , Assmus Birgit , Urbich Carmen , Zeiher Andreas M. , Dimmeler Stefanie
Published: June 2011

Abstract
—Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-α (TNF-α)–induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-α (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-α on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-α induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-α–induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9–induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.
Fluid Flow Releases Fibroblast Growth Factor-2 From Human Aortic Smooth Muscle Cells
Authors: Rhoads David N. , Eskin Suzanne G. , McIntire Larry V.
Published: June 2011

Abstract
—This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm
2
in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (
P
<0.01). A 50% increase in FGF-2 content versus control (
P
<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm
2
versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.
Homocysteine-Induced Inhibition of Endothelium-Dependent Relaxation in Rabbit Aorta
Authors: Lang Derek , Kredan Mahmud B. , Moat Stuart J. , Hussain Syed A. , Powell Catherine A. , Bellamy Michael F. , Powers Hilary J. , Lewis Malcolm J.
Published: June 2011

Abstract
—Hyperhomocysteinemia is associated with endothelial dysfunction, although its mechanism is unknown. Isometric tension recordings and lucigenin chemiluminescence were used to assess the effects of homocysteine exposure on endothelium-dependent and -independent relaxation in isolated rabbit aortic rings and superoxide anion (O
2
−
) production by cultured porcine aortic endothelial cells, respectively. Homocysteine (0.1 to 10 mmol/L) produced a significant (
P
<0.001) concentration- and time-dependent inhibition of endothelium-dependent relaxation in response to both acetylcholine and the calcium ionophore A23187. Only the intracellular O
2
−
scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L) significantly (
P
<0.001) inhibited the effect of homocysteine on acetylcholine- and A23187-induced relaxation. Incubation of porcine aortic endothelial cells with homocysteine (0.03 to 1 mmol/L for up to 72 hours) caused a significant (
P
<0.001) time-dependent increase in the O
2
−
released by these cells on the addition of Triton X-100 (1% [vol/vol]), with levels returning to values comparable to those of control cells at the 72-hour time point. These changes in O
2
−
levels were associated with a time-dependent increase in endothelial cell superoxide dismutase activity, becoming significant (
P
<0.001) after 72 hours. Furthermore, the homocysteine-induced increase in endothelial cell O
2
−
levels was completely inhibited (
P
<0.001) by the concomitant incubation with either Tiron (10 mmol/L), vitamin C (10 μmol/L), or vitamin E (10 μmol/L). These data suggest that the inhibitory effect of homocysteine on endothelium-dependent relaxation is due to an increase in the endothelial cell intracellular levels of O
2
−
and provide a possible mechanism for the endothelial dysfunction associated with hyperhomocysteinemia.
Homocysteine and Lipoprotein(a) Interact to Increase CAD Risk in Young Men and Women
Authors: Foody JoAnne Micale , Milberg John A. , Robinson Killian , Pearce Gregory L. , Jacobsen Donald W. , Sprecher Dennis L.
Published: June 2011

Abstract
—A biochemical link between homocysteine (tHcy) and lipoprotein(a) [Lp(a)] related to fibrin binding has been proposed. This hypothesis has not been specifically examined in human subjects. We sought to determine in a clinical setting whether these risk factors would interact to increase coronary artery disease (CAD) risk. We performed a cross-sectional analysis of 750 men and 403 women referred to a preventive cardiology clinic at the Cleveland Clinic Foundation, in whom baseline tHcy and Lp(a) data were available. Logistic regression after adjusting for standard cardiovascular risk factors was used to estimate the relative risk of CAD in patients with an Lp(a) ≥30 mg/dL and a tHcy ≥17 μmol/L. Neither isolated high tHcy (odds ratio [OR]=1.06,
P
=0.89) nor isolated high Lp(a) (OR=1.15,
P
=0.60) appeared to be associated with CAD in women. However, strong evidence of an association was seen when both risk factors were present (OR=4.83,
P
=0.003). Moreover, this increased risk showed evidence of an interactive effect beyond that attributable to either additive or multiplicative effects of tHcy and Lp(a) (
P
=0.03). In contrast, both elevated tHcy (OR=1.93,
P
=0.05) and elevated Lp(a) (OR=1.87,
P
=0.01) showed evidence of being independent risk factors for CAD in men. The presence of both risk factors in men did not appear to confer additional risk (OR=2.00,
P
=0.09), even though ORs as high as 12.4 were observed within specific age intervals. Consistent with prior studies, tHcy and Lp(a) are risk factors, either independently or in concert, for CAD in this clinical population. More significantly, we found evidence that when both risk factors were present in women, the associated risk was greater than what would be expected if the 2 risks were simply acting independently. The absence of such an interactive effect in men may be due to the confounding effects of age manifested as “survivor bias.” These clinical findings provide insights into the potential roles of both tHcy and Lp(a) in the pathogenesis of atherosclerosis.
Promoter Polymorphisms of Human Paraoxonase
PON1
Gene and Serum Paraoxonase Activities and Concentrations
Authors: Leviev Ilia , James Richard W.
Published: June 2011

Abstract
—Paraoxonase (PON) is a serum enzyme with a wide species distribution. It protects lipoproteins from toxic oxidative modifications and is an antiatherogenic mechanism of major potential. Activity levels of PON are major determinants of the protective function; consequently, factors that influence PON levels are of particular relevance. The present study has identified 3 polymorphisms in the promoter region of the human
PON1
gene. Cell transfection studies have revealed their variable impact on promoter activity, with up to 2-fold differences in reporter gene expression. Genotyping studies have established that the polymorphisms are frequent in the population, a finding that is consistent with a major impact on PON concentrations. The physiological relevance of the polymorphisms was underlined by showing that they are associated with highly significant differences in serum concentrations and activities of PON. The study thus firmly establishes a genetic basis for variations in serum PON levels and, consequently, serum PON activity. It is consistent with the suggestion that variations in a major antioxidant function of high density lipoprotein are, to an important degree, genetically determined.
Distinct Risk Profiles of Early and Advanced Atherosclerosis
Authors: Willeit Johann , Kiechl Stefan , Oberhollenzer Friedrich , Rungger Gregor , Egger Georg , Bonora Enzo , Mitterer Manfred , Muggeo Michele
Published: June 2011

Abstract
—Most epidemiological surveys on risk factors of atherosclerosis were cross-sectional in design and did not consider the existence of pathologically distinct processes. The Bruneck Study is a prospective survey in the general community (age range, 40 to 79 years). The baseline examination and first reevaluation were performed in the summers of 1990 and 1995 (participation, 92%; follow-up, 96%). Carotid atherosclerosis was monitored with high-resolution duplex ultrasound. Early (incidence and/or extension of nonstenotic lesions) and advanced (incidence and/or progression of stenosis >40%) stages of atherogenesis were differentiated. The risk profile of early atherogenesis consists of traditional risk factors, such as hypertension, hyperlipidemia, and cigarette smoking (pack-years), supplemented by a variety of less well-established risk conditions, including high body iron stores, hypothyroidism, microalbuminuria, and high alcohol consumption. In contrast, the risk profile of advanced atherogenesis includes markers of enhanced prothrombotic capacity, attenuated fibrinolysis, and clinical conditions known to interfere with coagulation: high fibrinogen, low antithrombin, factor V Leiden mutation, lipoprotein(a) >0.32 g/L, high platelet count, cigarette smoking, and diabetes. Hyperlipidemia and hypertension were of only minor relevance. These findings, along with the epidemiological features of advanced atherogenesis and emergence of an elevated fibrin turnover, suggest atherothrombosis to be a key mechanism in the development of advanced stenotic atherosclerosis. Supplementary 6-category logistic regression models illustrate the changing association between major risk predictors and atherosclerosis of increasing severity and substantiate appropriateness of the 40% threshold applied for the definition of advanced stenotic atherosclerosis. Atherosclerosis is a heterogeneous process that subsumes etiologically and epidemiologically distinct disease entities. The multifactorial etiology of atherosclerosis, which goes far beyond the traditional risk factors, has not yet achieved adequate attention in clinical practice and disease prevention.
Insulin Resistance Syndrome Predicts the Risk of Coronary Heart Disease and Stroke in Healthy Middle-Aged Men
Authors: Pyörälä Marja , Miettinen Heikki , Halonen Pirjo , Laakso Markku , Pyörälä Kalevi
Published: June 2011

Abstract
—The interpretation of conventional multivariate analyses concerning the relation of insulin to the risk of atherosclerotic disease is complex because of correlations of insulin with other risk factors. Therefore, we applied factor analysis to study the clustering of risk factors in the baseline data of the Helsinki Policemen Study (970 healthy men aged 34 to 64 years) and investigated whether these clusterings predict coronary heart disease (CHD) and stroke risk. Areas under the glucose and insulin response curves (AUC glucose and AUC insulin) were used to reflect glucose and insulin levels during oral glucose tolerance tests. During the 22-year follow-up, 164 men had a CHD event, and 70 men had a stroke. Factor analysis of 10 risk factor variables produced 3 underlying factors: insulin resistance factor (comprising body mass index, subscapular skinfold, AUC insulin, AUC glucose, maximal O
2
uptake, mean blood pressure, and triglycerides), lipid factor (cholesterol and triglycerides), and lifestyle factor (physical activity and smoking). In multivariate Cox models, the age-adjusted hazard ratio for insulin resistance factor during the 22-year follow-up was 1.28 (95% CI 1.10 to 1.50) with regard to CHD risk and 1.64 (95% CI 1.29 to 2.08) with regard to stroke risk. Lipid factor predicted the risk of CHD but not that of stroke, and lifestyle factor predicted a reduced CHD risk. Factor analysis including only 6 risk factor variables proposed to be central components of insulin resistance syndrome (body mass index, subscapular skinfold, AUC insulin, AUC glucose, mean blood pressure, and triglycerides) produced only a single insulin resistance factor that predicted the risk of CHD and stroke independently of other risk factors.
Factor VII Gene Polymorphism, Factor VII Levels, and Prevalent Cardiovascular Disease
Authors: Feng DaLi , Tofler Geoffrey H. , Larson Martin G. , O’Donnell Christopher J. , Lipinska Izabella , Schmitz Christian , Sutherland Patrice A. , Johnstone Michael T. , Muller James E. , D’Agostino Ralph B. , Levy Daniel , Lindpaintner Klaus
Published: June 2011

Abstract
—Elevated factor VII levels have been associated with increased cardiovascular risk in some studies. The arginine/glutamine (Arg/Gln) polymorphism of the factor VII gene has been previously shown to modify factor VII levels. However, the presence of a gene/environment interaction on factor VII levels or a link with cardiovascular disease (CVD) remains uncertain. We studied subjects from the Framingham Heart Study to determine (1) the extent to which this genetic polymorphism affects factor VII levels; (2) whether interactions exist between this polymorphism and environmental factors on factor VII levels; and (3) the association between the polymorphism and CVD. Genotype data and factor VII antigen levels were available in 1816 subjects. Factor VII levels differed significantly among genotypes in an additive fashion: Gln homozygous, 82.7±2.5%; heterozygous, 92.2±0.7%; and Arg homozygous, 100.5±0.4% (
P
<0.0001). The polymorphism was the strongest, single predictor of factor VII levels, explaining 7.7% of the total variance of factor VII levels, whereas other traditional risk factors combined explained an additional 11.5% of the variance. There was an interaction (
P
=0.02) between the genotype and total cholesterol on factor VII levels, such that the correlation coefficient and slope (factor VII level/total cholesterol) were greatest in Gln/Gln subjects. Among 3204 subjects characterized for genotype and CVD, there was no significant relationship between the genotype and CVD (
P
=0.12). In the Framingham Heart Study, the Arg/Gln polymorphism was significantly associated with factor VII antigen levels. The strength of the association suggests that genetic variation plays an important role in determining factor VII levels. However, despite being associated with factor VII levels, the Arg/Gln polymorphism was not associated with prevalent CVD.