bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Lp A-I and Lp A-I/A-II in Cholesteryl Ester Transfer Protein–Mediated Neutral Lipid Transfer
Authors: McPherson Ruth , Agnani Geneviève , Lau Paulina , Fruchart Jean-Charles , Edgar Alan D. , Marcel Yves L.
Published: June 2011

The two major subclasses of HDL contain apo A-I only (Lp A-I) or both apo A-I and apo A-II (Lp A-I/A-II). We have carried out experiments to quantify the participation of Lp A-I and Lp A-I/A-II in the neutral lipid transfer reaction in normal and hypertriglyceridemic subjects. Thirteen hypertriglyceridemic subjects were studied before and after fenofibrate therapy. Fenofibrate treatment resulted in decreases in total cholesterol, triglycerides (TG), and VLDL cholesterol of 19%, 48%, and 70%, respectively, and a 28% increase in HDL cholesterol, with no significant change in the proportion of Lp A-I and Lp A-I/A-II particles. The abundance of cholesteryl ester transfer protein (CETP) mRNA in peripheral adipose tissue decreased with treatment in four of five patients studied; however, no change occurred in plasma CETP mass. Using an isotopic transfer assay, we demonstrated that both Lp A-I and Lp A-I/A-II participated in the CE transfer reaction, with no change after fenofibrate therapy. This finding suggests that the marked increase in HDL cholesterol during fenofibrate therapy is due to normalization of plasma TG and hence decreased opportunity for mass transfer of lipid between HDL and TG-rich proteins in vivo. In this population of hypertriglyceridemic subjects, CETP was distributed in both the Lp A-I and Lp A-I/A-II subfractions of HDL, with preferential association with the smaller Lp A-I pool. In contrast, in nine normal subjects studied, negligible amounts of CETP were associated with Lp A-I/A-II. Nonetheless, the Lp A-I/A-II fraction of HDL contributed significantly to total CE mass transfer in normolipidemic plasma. Lp A-I/A-II is an efficient donor for CE transfer to TG-rich lipoproteins, and its low affinity for CETP may in fact facilitate neutral lipid transfer either by a shuttle mechanism or by formation of a ternary complex.
Effect of Dietary Fat Saturation on LDL Oxidation and Monocyte Adhesion to Human Endothelial Cells In Vitro
Authors: Mata P. , Alonso R. , Lopez-Farre A. , Ordovas J.M. , Lahoz C. , Garces C. , Caramelo C. , Codoceo R. , Blazquez E. , de Oya M.
Published: June 2011

Forty-two healthy men and women were subjected to four consecutive dietary periods differing in the fat content of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (n-6) [PUFA(n-6)] and (n-3) [PUFA(n-3)]. Plasma lipids, vitamin E, and in vitro LDL oxidation were examined during each period. Adhesion of human monocytes to cultured human endothelial cells was used as a functional test to identify differences in the biological properties of LDL from each dietary period. Consumption of an SFA-rich diet resulted in higher LDL cholesterol (4.06±0.85 mmol/L,
P
<.05) than did consumption of MUFA- (3.59±0.75 mmol/L), PUFA(n-6)– (3.44±0.77 mmol/L), or PUFA(n-3)– (3.31±0.8 mmol/L) rich diets. HDL cholesterol was lower during both PUFA-rich diets (1.24±0.28 and 1.27±0.28 mmol/L for n-6 and n-3, respectively) than during the SFA- (1.32±0.36 mmol/L) and MUFA- (1.32±0.34 mmol/L) rich diets. LDL resistance to copper-induced oxidation, expressed as lag time, was highest during the MUFA-rich diet (55.1±7.3 minutes) and lowest during the PUFA(n-3)– (45.3±7 minutes) and SFA- (45.3±6.4 minutes) rich diets. LDL induction of monocyte adhesion to endothelial cells was lower during the MUFA-rich diet than the other periods. The highest monocyte adhesion was obtained during the PUFA(n-3) and SFA dietary periods. In conclusion, an MUFA-rich diet benefits plasma lipid levels compared with an SFA-rich diet. Furthermore, this diet results in an increased resistance of LDL to oxidation and a lower rate of monocyte adhesion to endothelial cells than the other dietary fats examined.
A Cysteine-Containing Truncated Apo A-I Variant Associated With HDL Deficiency
Authors: Moriyama Kengo , Sasaki Jun , Takada Yoichi , Matsunaga Akira , Fukui Jun , Albers John J. , Arakawa Kikuo
Published: June 2011

We identified a 50-year-old Japanese woman with a novel mutation in the apolipoprotein (apo) A-I gene causing high-density lipoprotein (HDL) deficiency. The patient had extremely low HDL cholesterol and apo A-I levels (0.14 mmol/L and 0.8 mg/dL, respectively) but no evidence of coronary heart disease. However, she had bilateral xanthomas of the Achilles tendon, elbow, and knee joint as well as corneal opacities. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of serum followed by immunoblotting revealed that the patient's apo A-I had a lower molecular weight (24 000) than normal apo A-I. A partial gene duplication encompassing 23 nucleotides was found by DNA sequence analysis, resulting in a tandem repeat of bases 333 to 355 from the 5′ end of exon 4. This tandem repeat caused a frameshift mutation with premature termination after amino acid 207. The frameshift gives rise to a predicted protein sequence that contains two cysteines. We designated this mutant as apo A-I
Sasebo
. Apo A-I
Sasebo
formed heterodimers with apo A-II and apo E in the patient's plasma and was associated with both the low-density lipoprotein and HDL fractions. The patient's cholesterol esterification rate and lecithin-cholesterol acyltransferase activity were reduced to about 30% of normal, although specific enzyme activity was unaffected, suggesting that it remained functionally normal. In addition, cholesteryl ester transfer activity was reduced to about half of normal. Thus, apo A-I
Sasebo
was associated with complex derangements of lipoprotein metabolism.
Dietary and Genetic Effects on LDL Size Measures in Baboons
Authors: Singh Amareshwar T.K. , Rainwater David L. , Kammerer Candace M. , Sharp R. Mark , Poushesh Mahmood , Shelledy Wendy R. , VandeBerg John L.
Published: June 2011

Genetic and dietary effects on LDL phenotypes, including predominant LDL particle diameter, LDL size distribution, and non–HDL cholesterol and apoB concentrations, were investigated in 150 pedigreed baboons that are members of 19 sire groups. Baboons were fed a sequence of three defined diets differing in levels of fat and cholesterol. Increasing dietary fat had relatively little effect on two measures of LDL particle size. However, increasing the level of cholesterol in the diet resulted in larger increases of the predominant LDL particle diameters and in the proportion of stain on LDLs >28 nm. As expected, apoB and non–HDL cholesterol concentrations significantly increased when levels of dietary fat and cholesterol were increased. Correlations among the LDL phenotypes suggested that several different aspects of the LDL phenotype were captured by the four LDL measures across the three diets. Genetic effects indicated by sire group membership were significant both for expression of the LDL phenotypes and for response to changes in diet.
Peripheral Artery Disease and Cardiovascular Risk Factors in the Elderly
Authors: Curb J. David , Masaki Kamal , Rodriguez Beatriz L. , Abbott Robert D. , Burchfiel Cecil M. , Chen Randi , Petrovitch Helen , Sharp Dan , Yano Katsuhiko
Published: June 2011

Peripheral vascular disease as measured by the ankle/brachial blood pressure index (ABI) is associated with increased risk of mortality and morbidity. Few sources of data on the relationship of risk factors to ABI are available for the elderly, especially those >80 years of age, and minority populations. ABI measurements from the Honolulu Heart Program's fourth reexamination of 3450 ambulatory, elderly Japanese American men indicate that the prevalence of an abnormal ABI, defined as a ratio of <0.9, was 13.6%, increasing from 8.0% in those 71 to 74 years of age to 27.4% in those 85 to 93 years. Associations that were U or J shaped were present for a number of risk factors (higher rates of abnormality [ABI<0.9] in those in the lowest and highest risk factor quintiles) in a cross-sectional analysis. Risk factors measured at baseline were also predictive of an abnormal ABI 25 years later, even after adjustment for multiple risk factors. The odds ratio (OR) for an ABI<0.9 at the 80th percentile of cholesterol compared with that at the 20th percentile was 1.4; the OR for 1-hour postload glucose was 1.3, and for alcohol intake 1.2. The OR associated with hypertension was 1.8 and that for smoking, 2.9 (
P
<.05 for all ORs). These findings are consistent with ABI being a marker for generalized atherosclerotic disease in old and very old Japanese American men.
Induction of Hepatic Uptake of Lipoprotein(a) by Cholesterol-Derivatized Cluster Galactosides
Authors: Biessen Erik A.L. , Vietsch Helene , van Berkel Theo J.C.
Published: June 2011

We have previously developed triantennary galactosides [TG(4Å)C and TG(20Å)C] that lower cholesterol levels by inducing liver uptake of lipoproteins via galactose-recognizing hepatic receptors. In this study, we have investigated whether this strategy could also be applied to reduce elevated serum levels of the atherogenic lipoprotein(a) [Lp(a)]. Both TG(4Å)C and TG(20Å)C could be incorporated into Lp(a). Incorporation of these glycolipids induced a rapid clearance of Lp(a). Concomitantly, the hepatic uptake of
125
I-Lp(a) was enhanced from 4±1% to 80±4% of the injected dose for TG(4Å)C (
P
<.0001) and to 17±4% of the injected dose for TG(20Å)C (
P
<.006). TG(4Å)C was apparently more effective in accelerating the serum decay of
125
I-Lp(a), which may be caused by the higher hydrophobicity of this glycolipid relative to TG(20Å)C. The TG(4Å)C- and TG(20Å)C-induced stimulation of the serum decay and liver uptake of
125
I-Lp(a) could be significantly inhibited (>85%) by preinjection of
N
-acetyl-galactosamine (150 mg), indicating that galactose-recognizing receptors are involved in the liver uptake of the glycolipid/Lp(a) complexes. The TG(4Å)C-induced liver uptake of
125
I-Lp(a) could be ascribed mainly to Kupffer cells (76±7%), whereas the parenchymal liver cell was the major site for liver uptake of TG(20Å)C-laden
125
I-Lp(a) (55±12%). In conclusion, both TG(4Å)C and TG(20Å)C stimulate the catabolism of
125
I-Lp(a) by enhancing hepatic uptake. Because endocytosis of the substrate via galactose-recognizing receptors on Kupffer and parenchymal liver cells is followed by lysosomal degradation, we anticipate that both approaches for Lp(a) targeting may prove valuable as therapeutic modalities for lowering atherogenic levels of Lp(a).
Ethanol-Induced Redistribution of Cholesteryl Ester Transfer Protein (CETP) Between Lipoproteins
Authors: Hannuksela Minna L. , Rantala Maire , Kesäniemi Y. Antero , Savolainen Markku J.
Published: June 2011

AbstractSince alcohol drinking reduces the concentration and activity of plasma cholesteryl ester transfer protein (CETP), we investigated the effects of alcohol on its synthesis and secretion by perfusing rabbit livers for 4 hours in the absence or presence of ethanol. The quantity of CETP mRNA in the perfused livers did not differ between the control and ethanol (25 mmol/L or 50 mmol/L) perfusions. CETP activity was determined by incubating [3H]cholesteryl ester–labeled human LDL and unlabeled human HDL with the perfusion medium after removing the endogenous VLDL (secreted by the perfused liver) by ultracentrifugation. CETP activity in the perfusion medium increased at a linear rate that was not affected by ethanol. When the VLDL was removed by precipitation with polyethylene glycol or a heparin-Sepharose column instead of ultracentrifugation, practically no CETP activity was detected in the ethanol perfusions, whereas these procedures did not affect CETP activity in the control perfusions. Inhibition of ethanol oxidation by 4-methylpyrazole resulted in CETP activity similar to that of the controls. We conclude that ethanol does not affect the synthesis or secretion of CETP, but its oxidation may alter the distribution of CETP in lipoproteins. CETP seems to be present in VLDL as well as in HDL, and since VLDL is more rapidly catabolized than HDL, this may explain the low plasma CETP concentration associated with alcohol consumption.
Isolation and Characterization of Human Antioxidized LDL Autoantibodies
Authors: Mironova Marina , Virella G. , Lopes-Virella Maria F.
Published: June 2011

Abstract
Autoantibodies to oxidized LDL have been reported in normal subjects and in patients with arteriosclerosis, but their possible pathogenic role is not yet well defined. One important problem is the existence of contradictory data reported by different groups concerning the associations between antioxidized LDL autoantibodies and the presence or progression of arteriosclerotic lesions. Such contradictions led us to decide to isolate and characterize antioxidized LDL antibodies by affinity chromatography with the use of oxidized LDL cross-linked to Sepharose. Antioxidized LDL antibodies were isolated from selected serum samples obtained from eight subjects. Seven of them (six patients and one control subject) had high levels of antioxidized LDL antibody during screening. The other subject, a healthy volunteer, had a low level of antibody. All purified antibodies contained IgG (of subclasses 1 and 3) as the predominant isotype and were primarily specific for oxidized LDL but showed some cross-reactivity with malondialdehyde-modified LDL and native LDL. Two of the purified antibodies cross-reacted with cardiolipin. We determined average dissociation constants for the antioxidized LDL antibodies purified from five individuals, which varied between 2.4×10
−7
and 7.5×10
−7
mol/L, whereas the average dissociation constant of rabbit hyperimmune anti-LDL antibody was determined to be 2.7×10
−8
mol/L. In conclusion, we have purified human autoantibodies reactive with oxidized LDL that appear to be predominantly of moderate-to-low affinity and of variable cross-reactivity. The predominance of IgG1 and IgG3 antibodies is significant from the standpoint of potential pathogenicity, since these two subclasses activate the classic complement pathway system and have the highest binding affinities for Fcγ receptors on phagocytic cells.
Association of Localized Ca
2+
Gradients With Redistribution of Glycoprotein IIb-IIIa and F-actin in Activated Human Blood Platelets
Authors: Ariyoshi Hideo , Salzman Edwin W.
Published: June 2011

Abstract
We monitored the intracellular distribution of ionized free Ca
2+
concentration ([Ca
2+
]
i
) in individual human platelets by digital imaging fluorescence microscopy with fura 2 during platelet activation induced by surface contact or a soluble platelet agonist (thrombin). Contact of platelets with glass resulted in pseudopod formation and spreading, accompanied by a nonuniform rise in [Ca
2+
]
i
. The rise in [Ca
2+
]
i
was maximal during pseudopod formation. Locally elevated [Ca
2+
]
i
was frequently found in pseudopodia and at the edge and core of spread platelets. This pattern was faithfully duplicated by the local pattern of distribution of the cytoskeletal components F-actin, gelsolin, and surface glycoproteins (GP) IIb-IIIa but not by calmodulin. Platelets stimulated by thrombin also showed an inhomogeneous rise in [Ca
2+
]
i
, which was well correlated with the staining of F-actin and GPIIb-IIIa. Cytochalasin D, an inhibitor of actin polymerization, inhibited the inhomogeneous increase or redistribution of F-actin and GPIIb-IIIa but did not inhibit the rise in mean [Ca
2+
]
i
. These observations suggest that a localized change in [Ca
2+
]
i
may be associated with cytoskeletal reorganization and redistribution of GPIIb-IIIa in activated platelets.
Triiodothyronine Exerts a Major Pleiotropic Effect on Reverse Cholesterol Transport Phenotypes
Authors: Comuzzie Anthony G. , Blangero J. , Mahaney Michael C. , Sharp R. Mark , VandeBerg John L. , Stern Michael P. , MacCluer Jean W.
Published: June 2011

Abstract
The thyroid hormone triiodothyronine (T
3
) is known to be a potent mediator of
APOA1
gene expression. With the use of multivariate quantitative genetic analysis, we have assessed the magnitude of shared effects of T
3
on plasma concentrations of apolipoprotein AI (apo AI) and three related phenotypes: HDL-C, apo AII, and LpAI (which is a concentration of apo AI that contains HDL particles). Maximum likelihood techniques were used to simultaneously estimate mean effects and variance components in large, extended Mexican American families living in San Antonio, Tex. We found that T
3
accounted for 16%, 23%, 21%, and 37% of the additive genetic variance in HDL-C, apo AI, apo AII, and LpAI, respectively, while explaining virtually none of the random environmental variance in these phenotypes. T
3
also has a pronounced effect on the pairwise genetic correlations among the four phenotypes: After the pleiotropic effects of T
3
concentrations are controlled for, the genetic correlations are reduced by 6% in the case of HDL-C and apo AI and 97% for apo AII and LpAI. Thus, genes that influence T
3
have a significant effect on HDL-C, apo AI, apo AII, and LpAI and also on the correlations among these phenotypes.
Oral Estrogen Replacement Therapy in Postmenopausal Women Selectively Raises Levels and Production Rates of Lipoprotein A-I and Lowers Hepatic Lipase Activity Without Lowering the Fractional Catabolic Rate
Authors: Brinton Eliot A.
Published: June 2011

Abstract
To characterize the apolipoprotein (apo) subfraction specificity of the increase in high density lipoprotein (HDL) levels that is induced by oral estrogen and to explore the metabolic mechanisms thereof, six healthy, postmenopausal women were studied during each of two 5-week periods of a low-fat diet with and without ethinyl estradiol 0.05 mg/d. With estrogen, HDL cholesterol levels increased 36% (mean±SD, 47±15 versus 63±18 mg/dL, basal versus estrogen periods, respectively;
P
=.004), plasma levels of apo A-I increased by 27% (147±24 versus 186±34 mg/dL;
P
=.003), and apo A-II levels increased 17% (39±5 versus 46±4 mg/dL;
P
=.0003). Apo A-I mass in particles that contained apo A-I but no apo A-II (ie, Lp A-I) increased 66% (43±13 versus 71±17 mg/dL;
P
=.002) while the sum of apo A-I and apo A-II mass in particles with both apo A-I and apo A-II (ie, Lp A-I/A-II) showed only a nonsignificant 14% increase (144±23 versus 163±29 mg/dL;
P
=.12). In radioiodinated-protein turnover studies the production rate (PR) of each species changed in proportion to the change in its plasma concentration: Lp A-I PR increased 76% (4.7±1.8 versus 8.2±2.5 mg·kg
−1
·day
−1
;
P
=.001) while Lp A-I/A-II PR showed only a nonsignificant increase of 22% (14.1±1.8 versus 17.2±5.4 mg·kg
−1
·day
−1
;
P
=.2). Hepatic lipase (HL) activity in postheparin plasma decreased 66% with estrogen (10.4±4.3 versus 3.5±0.8 μmol·mL
−1
·h
−1
;
P
=.005) while lipoprotein lipase activity was unchanged (7.1±1.6 versus 7.6±1.6 μmol·mL
−1
·h
−1
;
P
>.2). Despite the large decrease in HL activity, the fractional catabolic rate of Lp A-I did not decrease (0.241±0.048 versus 0.258±0.066 pool/d;
P
=.2), nor was that of Lp A-I/A-II lessened (0.221±0.030 versus 0.233±0.053 pool/d;
P
>.2). Thus, oral estrogen replacement therapy has a novel and potentially antiatherogenic effect: a large and selective increase in Lp A-I levels. The metabolic mechanism of the estrogen-induced increase in Lp A-I concentration appears to be the increase in its PR. Although the ability of estrogen to suppress HL activity has been confirmed, surprisingly this decrease was not accompanied by any decrease in the fractional catabolic rate of Lp A-I or Lp A-I/A-II. This suggests that HL may not play a major role in regulating HDL protein catabolism during suppression of HL by estrogen.
Dose-Response Relationship Between Ischemic Heart Disease Mortality and Long-term Arsenic Exposure
Authors: Chen Chien-Jen , Chiou Hung-Yi , Chiang Ming-Hsi , Lin Li-Ju , Tai Tong-Yuan
Published: June 2011

Abstract
The cardiovascular effects of inorganic arsenic have been documented, but the dose-response relationship between ischemic heart disease (ISHD) and long-term arsenic exposure remains to be elucidated. Mortality rates from ISHD among residents in 60 villages of the area in Taiwan with endemic arseniasis from 1973 through 1986 were analyzed to examine their association with arsenic concentration in drinking water. Based on 1 355 915 person-years and 217 ISHD deaths, the cumulative ISHD mortalities from birth to age 79 years were 3.4%, 3.5%, 4.7%, and 6.6%, respectively, for residents who lived in villages in which the median arsenic concentrations in drinking water were <0.1, 0.1 to 0.34, 0.35 to 0.59, and ≥0.6 mg/L. A cohort of 263 patients affected with blackfoot disease (BFD), a unique arsenic-related peripheral vascular disease, and 2293 non-BFD residents in the endemic area of arseniasis were recruited and followed up for an average period of 5.0 years. There was a monotonous biological gradient relationship between cumulative arsenic exposure through drinking artesian well water and ISHD mortality. The relative risks were 2.5, 4.0, and 6.5, respectively, for those who had a cumulative arsenic exposure of 0.1 to 9.9, 10.0 to 19.9, and ≥20.0 mg/L-years compared with those without the arsenic exposure after adjustment for age, sex, cigarette smoking, body mass index, serum cholesterol and triglyceride levels, and disease status for hypertension and diabetes through proportional-hazards regression analysis. BFD patients were found to have a significantly higher ISHD mortality than non-BFD residents, showing a multivariate-adjusted relative risk of 2.5 (95% CI, 1.1 to 5.4).
Triple Drug Immunosuppression Significantly Reduces Aortic Allograft Arteriosclerosis in the Rat
Authors: Lemström Karl B. , Räisänen-Sokolowski Anne K. , Häyry Pekka J. , Koskinen Petri K.
Published: June 2011

AbstractWe evaluated the effect of triple drug immunosuppression (cyclosporine A 10 mg·kg−1·d−1, methylprednisolone 0.5 mg·kg−1·d−1, and azathioprine 2 mg·kg−1·d−1) on the development of allograft arteriosclerosis (chronic rejection). The recipients of rat aortic allografts from the DA (AG-B4, RT1a) to the WF (AG-B2, RT1u) strain were either treated with triple drug immunosuppression (n=23) or left untreated (n=23) and used as controls. The grafts were removed 7, 14, 30, 90, and 180 days after transplantation, and vascular wall changes were evaluated by quantitative histology, [3H]thymidine autoradiography, and immunohistochemistry. Nonimmunosuppressed aortic allografts developed progressive arteriosclerotic alterations 1 to 6 months after transplantation that were virtually identical to those observed during chronic rejection in human cardiac allografts. Linear regression analysis revealed that triple drug immunosuppression with clinically relevant dosages of drugs significantly reduced intimal thickening (r=.69 versusr=.88,P<.05). Concomitantly, there was a marked reduction in the number of inflammatory cells (P<.01) and their rate of proliferation (P<.025) in the allograft adventitia during the period of acute inflammation (30 days after transplantation). Immunohistochemistry revealed that the number of helper T cells (W3/25) and monocyte/macrophages (OX42) but not cytotoxic T cells (OX8) or natural killer cells (3.2.3) was significantly (P<.05) reduced. The number of adventitial cells expressing interleukin-2 receptor (CD25) (P<.05), MHC class II (OX6) (P<.05), and leukocyte function–associated antigen–1 α-chain (CD11a) (P<.025) was also significantly reduced at 30 days. Triple drug immunosuppression downregulated the induction of MHC class II and intercellular adhesion molecule–1 on the graft endothelium but had no significant effect on the number of subendothelial inflammatory cells. In addition, [3H]thymidine autoradiography demonstrated that triple drug immunosuppression significantly reduced the rate of cell proliferation in the media, composed of smooth muscle cells, 30 and 90 days after transplantation. Thus, triple drug immunosuppression efficiently reduced the development of allograft arteriosclerosis by downregulating the inflammatory response and the level of immune activation in the allograft adventitia during the acute rejection period, resulting in diminished intimal thickening of the graft in the long run. These results support the concept that allograft arteriosclerosis is due to or at least initiated by immune injury of the graft.
Purification, Properties, Sequencing, and Cloning of a Lipoprotein-Associated, Serine-Dependent Phospholipase Involved in the Oxidative Modification of Low-Density Lipoproteins
Authors: Tew David G. , Southan Christopher , Rice Simon Q.J. , Lawrence G. Mark P. , Li Haodong , Boyd Helen F. , Moores Kitty , Gloger Israel S. , Macphee Colin H.
Published: June 2011

Abstract
A novel LDL-associated phospholipase A
2
(LDL-PLA
2
) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. V
max
and
K
m
for the purified enzyme are 170 μmol·min
−1
·mg
−1
and 12 μmol/L, respectively. Extensive peptide sequence from LDL-PLA
2
facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl
p
-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca
2+
, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA
2
have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA
2
before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA
2
.
Defective Thrombin-Induced Calcium Changes and Aggregation of Bernard-Soulier Platelets Are Not Associated With Deficient Moderate-Affinity Receptors
Authors: McNicol A. , Sutherland M. , Zou R. , Drouin J.
Published: June 2011

Abstract
Cloning of the moderate-affinity, serpentine thrombin receptor has helped clarify the mechanism of thrombin-induced platelet activation. Proteolytic cleavage by thrombin generates a new amino terminal that autostimulates the receptor, leading to activation of multiple signaling pathways and the platelet response. The function of other thrombin receptors, such as high-affinity glycoprotein Ib (GPIb), on platelets and their relationships to the moderate-affinity receptor remain unclear. The present study examined the role of the moderate-affinity thrombin receptor in Bernard-Soulier syndrome (BSS) platelets, which contain low amounts of GPIb. Platelets from four BSS subjects displayed normal aggregation profiles and cytosolic calcium changes in response to moderate or high concentrations of thrombin. In contrast, the BSS platelet aggregation response was delayed and calcium changes were absent in response to low thrombin concentrations. Platelets from an asymptomatic BSS heterozygote displayed an activation profile similar to those of control individuals. Specific activation of the moderate-affinity receptor by a synthetic peptide caused similar aggregation in platelets from all individuals. The synthetic peptide also elicited calcium responses in BSS platelets. Platelets from the BSS subjects and from an individual with the May-Hegglin anomaly showed increased expression of the moderate-affinity thrombin receptor by flow-cytometric analyses. These results suggest that BSS platelets possess high levels of a functional moderate-affinity thrombin receptor, probably due to large platelet size, and provide indirect evidence that a high-affinity thrombin receptor is associated with GPIb.
The Mechanism of Inhibition of Factor III (Thromboplastin) Activity by Apolipoprotein B-100
Authors: Ettelaie Camille , James Nicola J. , Wilbourn Barry , Adam Jacqueline M. , Naseem Khalid M. , Bruckdorfer K. Richard
Published: June 2011

Abstract
Factor III (thromboplastin) activity is inhibited by apoB-100, but the mechanism of inhibition is unknown. By examining the effect of purified apoB-100 on factor III activity, we showed that apoB-100 can inhibit factor III via a different mechanism from that caused by the tissue-factor pathway–inhibitor, which is mainly carried on the surface of lipoproteins. Although the presence of calcium ions and factors X and VII may enhance the rate of inhibition, they are not a prerequisite for the inhibition of factor III by apoB-100. In addition, by investigating the changes in the UV spectra of apoB-100 on interaction with factor III and factors X and VII and by assigning the shifts in absorption spectra to particular amino acids, we showed that these interactions involve negative and positive residues within these proteins. By following the rates of interactions between apoB-100 and either factors III, X, or VII, a two-step mechanism for the inhibition process involving factors X and VII was postulated. In this mechanism, the primary interaction of apoB-100 with factor III is followed by a rate-limiting step that can be accelerated by the presence of either factor X or VII and leads to the inhibition of factor III. Furthermore, a computer-based analysis of the sequences of factor III revealed a possible binding site for apoB-100.
Lipoprotein(a) Inhibits Collagen-Induced Aggregation of Thrombocytes
Authors: Gries Anna , Gries Maria , Wurm Helmut , Kenner Thomas , Ijsseldijk Martin , Sixma Jan J. , Kostner Gert M.
Published: June 2011

Abstract
Lipoprotein(a) [Lp(a)] is known to interact with human platelets in vitro. In the present study the effect of physiological concentrations of Lp(a) on platelet aggregation was studied. Freshly prepared gel-filtered platelets from healthy donors were incubated for 30 minutes at 37°C with various concentrations of Lp(a); aggregation was triggered with ADP, thrombin, and collagen. Control incubations were performed with Tyrode’s solution or LDL. Thrombin- and ADP-triggered aggregations were only slightly influenced by Lp(a), but aggregation of platelets stimulated with collagen (4 μg/mL) was markedly inhibited. Measurable effects occurred at low concentrations (0.05 mg/mL) of total Lp(a); at 0.5 mg/mL, maximum aggregation of platelets was inhibited by 54±20%, and the aggregation rate was attenuated by 47±19% compared with platelets incubated with Tyrode’s solution. Preincubation of collagen (4 μg/mL) with Lp(a) yielded similar results. The effect of Lp(a) on platelet aggregation was accompanied by a significant reduction of serotonin release and TXA
2
formation. Higher concentrations of collagen (≥10 μg/mL) caused the inhibitory effect of Lp(a) on collagen-induced aggregation to disappear. In contrast, incubation of platelets with 5 mg/mL LDL led to a significant increase of aggregation rate, maximum aggregation, serotonin release, and formation of TXA
2
when aggregation was induced with 4 μg/mL collagen. In an adhesion assay using fresh whole blood, which mimics the in vivo situation of vessel injury, Lp(a) reduced platelet adhesion at shear rates of 300 and 1600/s by 22.6% and 11.6%, respectively. In addition, Lp(a) reduced the size of platelet aggregates significantly (up to 63%); this reduction was more distinct at the higher shear rate. Unlike LDL, Lp(a) is not a proaggregatory lipoprotein; rather, collagen-triggered aggregation in vitro is attenuated by Lp(a).
Complement-Induced Release of Monocyte Chemotactic Protein-1 From Human Smooth Muscle Cells
Authors: Torzewski Jan , Oldroyd Rodney , Lachmann Peter , Fitzsimmons Colin , Proudfoot Diane , Bowyer David
Published: June 2011

AbstractIncreasing evidence suggests that complement activation might represent an important mechanism in early atherogenesis. Thus, complement components, in particular the membrane attack complex (MAC) C5b-9(m), have been isolated from human atherosclerotic lesions. Furthermore, complement activation is known to occur in atherosclerotic lesions induced in experimental animals, and the severity of cholesterol-induced plaques is markedly reduced in complement-deficient animals. During atherogenesis monocytes are recruited into the arterial wall, and a potent chemoattractant for monocytes, monocyte chemotactic protein-1 (MCP-1), is expressed by vascular smooth muscle cells (SMCs). We hypothesized that generation of MACs on SMCs during the activation of complement might lead to the release of MCP-1 and hence to monocyte recruitment. In this study, MACs were generated on human SMCs in vitro by sequential addition of the purified complement components C5b6, C7, C8, and C9. The supernatant of the culture was chemotactic for freshly isolated peripheral blood monocytes in a modified Boyden chamber. The chemotactic activity of the supernatant was abolished by anti–MCP-1 blocking antibodies but not by an isotype-matched antibody against an irrelevant antigen. The release of chemotactic activity was dependent on the dose of MAC formed on SMCs and was demonstrated within 10 minutes of exposure of the cells. The data support the hypothesis that complement-mediated release of MCP-1 from SMCs might be important in the recruitment of monocytes into the developing atherosclerotic lesion and could be an important initiating event in atherogenesis.
Low Whole-Blood
S
-Adenosylmethionine and Correlation Between 5-Methyltetrahydrofolate and Homocysteine in Coronary Artery Disease
Authors: Loehrer Franziska M.T. , Angst Christian P. , Haefeli Walter E. , Jordan Paul P. , Ritz Rudolf , Fowler Brian
Published: June 2011

Abstract
Mild elevation of plasma homocysteine is an independent risk factor for vascular disease. We studied the role of 5-methyltetrahydrofolate (5-MTHF), the folate form directly involved in homocysteine metabolism, in contrast to previous studies, which used total folate measurements, in 70 coronary artery disease (CAD) patients and control subjects. We also measured
S-
adenosylmethionine (SAM), which controls the activity of critical enzymes of homocysteine metabolism. Fasting plasma total homocysteine was elevated (>12.4 μmol/L for women, >13.3 μmol/L for men) in 17% of patients, in accordance with earlier studies. These patients showed lower 5-MTHF (12.4±1.0 nmol/L, mean±SD) than control subjects (24.2±15.0,
P
<.001), and there was a clear correlation (multiple linear regression analysis:
P
=.002) of this relevant form of folate with homocysteine. However, 37% of the normohomocysteinemic patients also revealed similarly low 5-MTHF levels, suggesting that a decrease of 5-MTHF does not necessarily cause hyperhomocysteinemia. SAM was significantly decreased in patients (1.4±0.4 μmol/L) compared with control subjects (1.8±0.3,
P
<.001) but was not correlated to homocysteine or 5-MTHF. The correlation between homocysteine and 5-MTHF that was found in CAD patients but not in control subjects confirms the direct relationship between these compounds in vivo. The new finding of low SAM in patients demands further studies, since it might indicate that low levels pose risk and that SAM might be a protective factor against the development of CAD.
Apolipoprotein A-II Influences the Substrate Properties of Human HDL
2
and HDL
3
for Hepatic Lipase
Authors: Mowri Hiro-Omi , Patsch Josef R. , Gotto Antonio M. , Patsch Wolfgang
Published: June 2011

Abstract
Hepatic lipase has a demonstrated dual role in plasma lipid transport in that it participates in the removal of remnants of triglyceride-rich lipoproteins from the circulation and in the metabolism of plasma HDL. The study presented here investigated the substrate properties for hepatic lipase of HDL differing in density and apolipoprotein (apo) composition. Rates of fatty acid liberation were twofold higher in HDL
2
compared with the respective HDL
3
subspecies. Within each density class, enzyme-catalyzed fatty acid release was nearly twofold higher from HDL containing apoA-II compared with HDL devoid of apoA-II. When native HDL
3
devoid of apoA-II was reconstituted with dimeric apoA-II in vitro, rates of fatty acid liberation in reconstituted particles were similar to those in native HDL
3
containing apoA-II. HDL containing apoA-II competed more effectively with small VLDL for binding of hepatic lipase than HDL devoid of apoA-II. HDL
3
, particularly apoA-II–containing HDL
3
, reduced lipolysis of triglyceride and total fatty acid liberation in small VLDL. We conclude that the substrate properties of HDLs for hepatic lipase are influenced by both their size and apoA-II content. Moreover, size as well as apoA-II content may indirectly affect remnant clearance.
Measurement of the Free Form of TFPI Antigen in Hyperlipidemia
Authors: Kokawa Toshinori , Enjyoji Kei-ichi , Kumeda Kousuke , Kamikubo Yu-ichi , Harada-Shiba Mariko , Koh Hideki , Tsushima Motoo , Yamamoto Akira , Kato Hisao
Published: June 2011

Abstract
Tissue factor pathway inhibitor (TFPI), a protease with three tandem Kunitz-type (K1, K2, and K3) domains, inhibits the initial reaction of the TF-mediated coagulation pathway. TFPI occurs in a free and a lipoprotein-associated form in plasma as well as an endothelial cell–associated form on vascular walls. In a previous study we had demonstrated that free-form TFPI activity was lower in hyperlipidemic patients. In the present study we established a new enzyme immunoassay method for measuring free-form TFPI antigen; this new method uses a monoclonal antibody that recognizes the K3 domain of free-form TFPI but not lipoprotein-associated TFPI. Free-form TFPI antigen was significantly lower in hyperlipidemic patients compared with those in normolipidemic individuals. We applied this new method to measure the amount of endothelial cell–associated TFPI, which can be released by heparin injection, as “free-form TFPI.” We found that free-form TFPI antigen in plasma was positively correlated with the endothelial cell–associated form. These results indicate that both of these forms of TFPI are in equilibrium in vivo and that our new method can be used for assessing changes in the levels of endothelial cell–associated TFPI antigen and, hence, for assessing thrombotic tendencies in various disease states.
A Single Nucleotide Insertion in Codon 317 of the
CD36
Gene Leads to CD36 Deficiency
Authors: Kashiwagi Hirokazu , Tomiyama Yoshiaki , Nozaki Shuichi , Honda Shigenori , Kosugi Satoru , Shiraga Masamichi , Nakagawa Tsutomu , Nagao Nobuo , Kanakura Yuzuru , Kurata Yoshiyuki , Matsuzawa Yuji
Published: June 2011

CD36 is a multifunctional integral-membrane glycoprotein that acts as a receptor for thrombospondin, collagen, long-chain fatty acids, and oxidized LDL. Platelet CD36 deficiency can be divided into two groups. In type I, neither platelets nor monocytes/macrophages express CD36; in type II, monocytes/macrophages express CD36 but platelets do not. Two known mutations cause CD36 deficiency, ie, a
478
C→T substitution in codon 90 (proline90→serine) and a dinucleotide deletion at nucleotide 539 in codon 110. In this study we investigated a type I Japanese subject (A.T.) and identified a new mutation, a single nucleotide insertion at nucleotide 1159 in codon 317. This mutation leads to a frameshift and the appearance of a premature stop codon.
CD36
gene analysis indicated that A.T. was a compound heterozygote for a dinucleotide deletion at nucleotide 539 and the single nucleotide insertion at nucleotide 1159. RNase protection studies suggested that the new mutation as well as the dinucleotide deletion led to a marked reduction in the level of
CD36
mRNA in her macrophages. However, the new mutation could be detected in macrophage but not platelet
CD36
mRNA. These data suggest that the allele having the single nucleotide insertion in this subject has an additional abnormality that results in the absence of the mutated
CD36
mRNA in platelets.
Gemfibrozil Stimulates Apolipoprotein A-I Synthesis and Secretion by Stabilization of mRNA Transcripts in Human Hepatoblastoma Cell Line (Hep G2)
Authors: Jin Fu-You , Kamanna Vaijinath S. , Chuang Mei-Yu , Morgan Kengathevy , Kashyap Moti L.
Published: June 2011

Gemfibrozil is a widely used drug that elevates plasma HDL and lowers triglycerides and LDL. The mechanism of action of this pharmacological agent on HDL metabolism is not established. Since the liver is the major organ involved in HDL production and removal, we assessed the effect of gemfibrozil on the modulation of apoA-I (a major protein of HDL)–containing particles by a human hepatoblastoma cell line (Hep G2). Incubation of Hep G2 cells with gemfibrozil resulted in the following statistically significant findings: (1) increased accumulation of apoA-I in the medium without affecting uptake of radiolabeled HDL-protein or HDL–apoA-I; (2) accelerated incorporation of [
3
H]leucine and [
35
S]methionine into apoA-I; (3) equivalent increases in [
3
H]leucine incorporation into HDL particles without and with apoA-II (LpA-I and LpA-I+A-II, respectively); (4) equal efflux of fibroblast cholesterol by harvested LpA-I and LpA-I+A-II particles; (5) increased steady state apoA-I mRNA without affecting apoA-I transcription; and (6) increased apoA-I mRNA half-life (2.2-fold). These data indicate that gemfibrozil stabilizes apoA-I mRNA transcripts, resulting in increased translation of functional apoA-I–containing particles capable of effluxing cellular cholesterol, thus defining a major mechanism by which gemfibrozil increases HDL.
Effect of Estrogen and Progesterone on the Expression of Hepatic and Extrahepatic Sterol 27-Hydroxylase in Baboons (
Papio
sp)
Authors: Kushwaha Rampratap S. , Guntupalli Bharathi , Jackson Evelyn M. , McGill Henry C.
Published: June 2011

Sterol 27-hydroxylase plays an important role in cholesterol metabolism in hepatic and extrahepatic tissues. To determine whether female sex steroid hormones influence its expression, we measured plasma and hepatic 27-hydroxycholesterol, hepatic mRNA levels, activity of sterol 27-hydroxylase, and adrenal mRNA levels of this enzyme in baboons (n=6 per group) treated with placebo, estrogen, estrogen + progesterone, and progesterone. We also measured hepatic cholesterol concentration and hepatic acyl coenzyme A:cholesterol acyltransferase (ACAT) activity to determine their relationship with hepatic sterol 27-hydroxylase activity. Plasma 27-hydroxycholesterol concentration was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (
P
=.090) and LDL (
P
=.026) cholesterol concentrations. Similarly, hepatic sterol 27-hydroxylase activity was increased by estrogen and estrogen + progesterone and was negatively correlated with plasma (
P
=.056) and LDL (
P
=.052) cholesterol concentrations but was positively correlated with hepatic and plasma 27-hydroxycholesterol concentrations (
P
<.001). Hepatic ACAT activity was increased by progesterone (
P
<.004) and was positively correlated with plasma (
P
=.002) and LDL (
P
=.009) cholesterol concentrations but was negatively correlated with hepatic sterol 27-hydroxylase activity (
P
=.035). Hepatic and adrenal gland mRNA levels for sterol 27-hydroxylase were increased by estrogen alone or in combination with progesterone (
P
<.05). Hepatic sterol 27-hydroxylase activity was positively correlated with hepatic mRNA levels (
P
<.001), an observation suggesting that estrogen increases the activity of sterol 27-hydroxylase by increasing its synthesis. Hepatic cholesterol concentration was not influenced by the hormone treatment. These observations suggest that estrogen alone or in combination with progesterone increases the synthesis of sterol 27-hydroxylase in hepatic and extrahepatic tissues, and the increased activity of hepatic sterol 27-hydroxylase resulting from the increased synthesis is associated with a hypolipidemic effect on plasma LDL levels. Furthermore, progesterone alone increases the hepatic ACAT activity, but given in combination with estrogen progesterone does not have the same effect on hepatic ACAT activity. The effect of estrogen on hepatic ACAT activity may be mediated by sterol 27-hydroxylase and its effect on cholesterol metabolism (decreased cholesterol synthesis and increased output of cholesterol in the bile) in liver.
Both Lipolysis and Hepatic Uptake of VLDL Are Impaired in Transgenic Mice Coexpressing Human Apolipoprotein E*3Leiden and Human Apolipoprotein C1
Authors: Jong Miek C. , Dahlmans Vivian E.H. , van Gorp Patrick J.J. , Breuer Marco L. , Mol Marc J.T.M. , van der Zee André , Frants Rune R. , Hofker Marten H. , Havekes Louis M.
Published: June 2011

Transgenic mice overexpressing human
APOE*
3Leiden are highly susceptible to diet-induced hyperlipoproteinemia and atherosclerosis due to a defect in hepatic uptake of remnant lipoproteins. In addition to the human
APOE
*3Leiden gene, these mice carry the human
APOC1
gene (
APOE
*3Leiden-
C1
). To investigate the possible effect of simultaneous expression of the human
APOC1
gene, we examined the phenotypic expression in these
APOE
*3Leiden-
C1
mice in relation to transgenic mice expressing the
APOE
*3Leiden gene without the
APOC1
gene (
APOE
*3Leiden-HCR).
APOE
*3Leiden-
C1
and
APOE
*3Leiden-HCR mice had comparable liver expression for the
APOE
*3Leiden transgene and high total cholesterol levels on a sucrose-based diet compared with control mice (4.3 and 4.3 versus 2.1 mmol/L). In addition, on this diet
APOE
*3Leiden-
C1
mice displayed significantly higher serum triglyceride levels than
APOE
*3Leiden-HCR mice and control mice (4.4 versus 0.6 and 0.2 mmol/L). Elevated triglyceride and cholesterol levels were mainly in the VLDL-sized lipoproteins. In vivo turnover studies with endogenously triglyceride-labeled VLDL showed a reduced VLDL triglyceride fractional catabolic rate for
APOE
*3Leiden-
C1
and
APOE
*3Leiden-HCR mice compared with control mice (3.5 and 11.0 versus 20.4 pools per hour). To study whether the difference in fractional catabolic rates between the two transgenic strains was due to an inhibiting effect of apoC1 on the extrahepatic lipolysis or hepatic-mediated uptake of VLDL, turnover experiments were performed in functionally hepatectomized mice. Strikingly, both
APOE
*3Leiden-
C1
and
APOE
*3Leiden-HCR mice showed a decreased lipolytic rate of VLDL triglyceride in the extrahepatic circulation compared with control mice (1.5 and 1.8 versus 6.3 pools per hour). We conclude that next to an impaired hepatic uptake, overexpression of the
APOE
*3Leiden gene influences the extrahepatic lipolysis of VLDL triglycerides, whereas simultaneous overexpression of the
APOC1
gene leads to a further decrease in hepatic clearance of VLDL.