bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidized LDL Binds to CD36 on Human Monocyte-Derived Macrophages and Transfected Cell Lines
Authors: Nicholson Andrew C. , Frieda S. , Pearce A. , Silverstein Roy L.
Published: June 2011

AbstractAccumulating evidence strongly implicates oxidized LDL (Ox-LDL) in the pathogenesis of atherosclerosis. Several receptors have been identified that bind and internalize Ox-LDL, but their relative importance in vivo is unclear. CD36 is an 88-kD transmembrane glycoprotein expressed on monocytes/macrophages, platelets, and microvascular endothelium that has been implicated as a putative receptor for Ox-LDL. We demonstrate that an anti-CD36 monoclonal antibody inhibited 50% of the specific binding and 26% of the specific degradation of Ox-LDL by human monocyte–derived macrophages. To characterize more completely this binding we evaluated interactions between CD36 and Ox-LDL in murine NIH-3T3 cells stably transfected with human CD36 cDNA. Ox-LDL bound to CD36-transfected 3T3 cells in a saturable manner. Specific binding, internalization, and degradation of Ox-LDL were increased fourfold in CD36-transfected cell lines compared with 3T3 cells transfected with vector alone. Binding of Ox-LDL to CD36-transfected 3T3 cells was inhibited by a panel of anti-CD36 antibodies and by soluble CD36 but not by thrombospondin. Specificity of binding was demonstrated by the equivalent binding of LDL and acetylated LDL to control and CD36-transfected 3T3 cells. The epitope or epitopes on Ox-LDL recognized by CD36 are undefined. Two observations suggest that CD36 recognizes a lipid moiety or that the lipid portion of the lipoprotein is essential for apoprotein recognition. The first is that the increased binding of Ox-LDL to CD36-transfected 3T3 cells is abrogated by delipidation of the lipoprotein, and the second is that oleic acid competes for the binding of Ox-LDL to CD36-transfected 3T3 cells. These data demonstrate that CD36 functions as an Ox-LDL receptor and suggest that CD36 may play a functional role in lipid accumulation by human macrophages and subsequent foam cell development during atherosclerosis.
Apolipoprotein A-I–Mediated Efflux of Sterols From Oxidized LDL–Loaded Macrophages
Authors: Kritharides Leonard , Jessup Wendy , Mander Erin L. , Dean Roger T.
Published: June 2011

Abstract
Although oxidized low-density lipoprotein (OxLDL) can accumulate in macrophages in vitro, generating cholesterol-loaded cells, little attention has been paid to the capacity of such macrophages loaded with OxLDL to export cholesterol and oxidized sterol moieties. In vitro lipid-loaded cells were generated by incubating primary cultures of mouse peritoneal macrophages with acetylated LDL (AcLDL) or OxLDL for 24 hours. The cellular content of native cholesterol, individual cholesteryl esters, and 7-ketocholesterol was determined by high-performance liquid chromatography. These cells were then incubated with medium containing apolipoprotein (apo) A-I and albumin or albumin alone for up to 24 hours; cholesterol and oxidized sterol efflux were measured both in terms of intracellular depletion and extracellular accumulation. Macrophages loaded with AcLDL accumulated cholesterol and large quantities of cholesteryl esters, whereas OxLDL-loaded cells accumulated cholesterol, a number of oxidized compounds (predominantly 7-ketocholesterol), and a relatively small quantity of cholesteryl esters. AcLDL-derived cells released approximately 50% of their total cholesterol (unesterified and esterified) to apo A-I–containing medium over 24 hours in the form of unesterified cholesterol, whereas OxLDL-derived cells released approximately 30% of their total cholesterol and 7% of their total content of 7-ketocholesterol over the same period. There was minimal efflux of any sterol in the absence of apo A-I. The proportions of cholesterol and 7-ketocholesterol released by either AcLDL- or OxLDL-loaded cells were not reduced by inhibiting cellular acyl-CoA:cholesterol acyl transferase using Sandoz 58-035, despite substantial alterations in the proportions of both free cholesterol and (in OxLDL-loaded cells) free 7-ketocholesterol in these cells. Furthermore, the subcellular distributions of both cholesterol and 7-ketocholesterol in individual subcellular organelle fractions were identical to that of free cholesterol in nonloaded cells, indicating that these sterols in OxLDL-loaded cells are not selectively sequestered in lysosomes. 7-Ketocholesterol is released much less efficiently than cholesterol from OxLDL-loaded cells. In addition, OxLDL-loaded cells release cholesterol less efficiently than do cells derived from AcLDL. It is possible that this impairment of efflux from OxLDL-loaded cells influences the generation and persistence of the foam cell phenotype in vivo and may therefore contribute to the atherogenicity of OxLDL.
Apolipoprotein A-II Production Rate Is a Major Factor Regulating the Distribution of Apolipoprotein A-I Among HDL Subclasses LpA-I and LpA-I:A-II in Normolipidemic Humans
Authors: Ikewaki Katsunori , Zech Loren A. , Kindt Marie , Brewer H. Bryan , Rader Daniel J.
Published: June 2011

Abstract
HDLs are heterogeneous in their apolipoprotein composition. Apolipoprotein (apo) A-I and apoA-II are the major proteins found in HDL and form the two major HDL subclasses: those that contain only apoA-I (LpA-I) and those that contain both apoA-I and apoA-II (LpA-I:A-II). Substantial evidence indicates that these two subclasses differ in their in vivo metabolism and effect on atherosclerosis, with LpA-I the more specifically protective subfraction against atherosclerosis. The purpose of this study was to investigate the effect of apoA-I and apoA-II production and catabolism on plasma LpA-I and LpA-I:A-II levels. Fifty normolipidemic subjects (those with HDL cholesterol levels in the top and bottom tenth percentiles were excluded) underwent kinetic studies with radiolabeled apoA-I and apoA-II, and the kinetic parameters of apoA-I and apoA-II were correlated with LpA-I and LpA-I:A-II levels. ApoA-I levels were strongly correlated with apoA-I residence times and less strongly correlated with apoA-I production rates. In contrast, apoA-II levels were correlated only with apoA-II production rates and not with apoA-II residence times. Levels of apoA-I in LpA-I were correlated with apoA-I residence times, whereas levels of apoA-I in LpA-I:A-II were correlated primarily with apoA-II production rates. The fraction of apoA-I in LpA-I was highly inversely correlated with apoA-II production rate (
r
=−.67,
P
<.001). In multiple regression analysis, apoA-II production rate was the most significant independent variable determining percent apoA-I in LpA-I among all the kinetic parameters. These results indicate that in normolipidemic individuals (1) apoA-I levels are regulated primarily by apoA-I catabolism and apoA-II levels by apoA-II production; (2) the rate of catabolism of apoA-I is an important factor determining LpA-I levels, while the rate of apoA-II production is the major determinant of the amount of apoA-I in LpA-I:A-II; and (3) the rate of apoA-II production is a major factor determining the distribution of apoA-I between LpA-I and LpA-I:A-II, thereby possibly modulating susceptibility to atherosclerosis in humans.
Evaluation of the Sialic Acid Content of LDL as a Marker of Coronary Calcification and Extracoronary Atherosclerosis in Asymptomatic Hypercholesterolemic Subjects
Authors: Chappey B. , Myara I. , Giral P. , Kerharo G. , Plainfosse M.C. , Levenson J. , Simon A. , Moatti N.
Published: June 2011

Abstract
Recent studies have shown that the sialic acid content of LDL isolated from patients with angiographically demonstrated advanced coronary atherosclerosis is lower than that of LDL isolated from healthy subjects. These observations raise the question as to whether LDL sialic acid content could be used as an early marker of atherosclerosis. We screened for carotid, aortic, and femoral plaques by ultrasonography and for coronary calcifications by ultrafast computed tomography in 160 hypercholesterolemic subjects free of cardiovascular disease to investigate the relation between LDL sialic acid content and the prevalence of these early atherosclerotic lesions. LDL sialic acid values varied from 19.6 to 46.6 nmol/mg LDL protein (33.9±4.4, mean±SD) in the whole population, but the distribution was very similar: (1) in subjects with no plaque (34.1±4.9) relative to those with one or several plaques at one (34.2±4.4), two (33.0±3.6), or three (34.8±3.4) different arterial sites; (2) in subjects with (33.9±3.7) and without (34.1±4.8) coronary calcification; and (3) in subjects with both extracoronary and coronary lesions (33.8±3.9) relative to those with no arterial lesions (34.2±4.5). LDL sialic acid content was not related to sex, age, body mass index, smoking, blood pressure, or serum total cholesterol and lipoprotein(a) levels but correlated negatively with serum triglyceride levels (
P
<.001). These results suggest that LDL sialic acid content is not a discriminant marker of early atherosclerosis in asymptomatic hypercholesterolemic subjects.
Effect of Glycation on the Properties of Lipoprotein(a)
Authors: Makino Kazuhiko , Furbee James W. , Scanu Angelo M. , Fless Gunther M.
Published: June 2011

Abstract
Lipoprotein(a) [Lp(a)] was glycated by incubation in vitro with glucose (0 to 200 mmol/L), and its properties were compared with native Lp(a) and native and glycated LDL. Glucose was incorporated into Lp(a) in proportions that mirrored the distribution of lysines between apolipoprotein (apo) B-100 and apo(a). Because the kringle IV domains of apo(a) are lysine poor, only 10% of glucose bound to apo(a), whereas 90% was attached to the apoB-100 of Lp(a). Approximately 3% of the lysines of both Lp(a) and LDL were modified, which is a level comparable with that observed in LDL isolated from diabetic individuals. Glucose uptake by Lp(a) and LDL was almost identical and was linear as a function of concentration and time. Glycation increased the negative charge of Lp(a) and LDL as monitored by electrophoresis and ion-exchange chromatography and also reduced the affinity of Lp(a) and LDL for heparin-Sepharose. Glycation did not affect the lysine-binding property of Lp(a) or generate measurable malondialdehyde oxidation adducts. The catabolism of glycated Lp(a) by human monocyte–derived macrophages (HMDMs), like that of native Lp(a), was largely LDL receptor independent. Both glycated Lp(a) and LDL were degraded at a comparatively faster rate and stimulated greater cholesteryl ester formation than their unmodified counterparts. However, the degradation rate of glycated Lp(a) was approximately four- to fivefold slower and its stimulation of cholesteryl ester formation was ninefold lower than that of either form of LDL. These results show that Lp(a) can be glycated nonenzymatically in vitro, that the incorporation of glucose is dependent on the distribution of lysines between apo(a) and apoB-100, and that glycation does not affect the lysine-binding properties of Lp(a). Furthermore, glycation produced modest increases in the degradation rate of Lp(a) and associated cholesteryl ester synthesis by HMDMs. Based on these data, glycation does not appear to significantly enhance the atherogenic potential of unmodified Lp(a).
Lipoprotein Lipase Synergizes With Interferon Gamma to Induce Macrophage Nitric Oxide Synthetase mRNA Expression and Nitric Oxide Production
Authors: Renier Geneviève , Lambert Anick
Published: June 2011

AbstractLipoprotein lipase (LPL) induces macrophage tumor necrosis factor-α (TNF-α) gene expression and protein secretion. Since TNF-α can increase interferon gamma (IFN-γ)–dependent nitric oxide (NO) production, we studied whether LPL may synergize with IFN-γ for the induction of macrophage NO production. Although ineffective by itself, LPL in combination with IFN-γ increasedl-arginine–dependent NO production in a dose-dependent manner. Preincubation of LPL with an anti-LPL neutralizing antibody totally suppressed this effect. Increased NO synthetase (NOS) mRNA expression was also observed after macrophage treatment with IFN-γ and LPL. Protein synthesis was required for the induction of NOS mRNA, and a TNF-α–mediated effect of LPL on NOS gene expression and NO production was observed. The ability of LPL to augment IFN-γ–dependent NOS mRNA expression was associated with an increase in the NOS gene transcriptional activity but not in the NOS mRNA stability. Finally, binding of nuclear proteins to the nuclear factor–κB– and TNF-α–responsive sequences of the macrophage NOS promotor was decreased by treatment of the cells by IFN-γ alone or in combination with LPL. These data provide evidence for a link between LPL and arginine metabolism in macrophages and further stress the role of LPL in the regulation of macrophage activation.
Bimodal Distribution of Cholesteryl Ester Transfer Protein Activities in Normotriglyceridemic Men With Low HDL Cholesterol Concentrations
Authors: Tato Federico , Vega Gloria Lena , Grundy Scott M.
Published: June 2011

Abstract
Increased plasma activities of cholesteryl ester transfer protein (CETP) theoretically could lower HDL cholesterol levels due to enhanced transfer of cholesteryl esters from HDL to apo B–containing lipoproteins. To determine whether high CETP activities are associated with isolated hypoalphalipoproteinemia, CETP activities were measured in 109 adult men with HDL cholesterol <35 mg/dL, plasma triglycerides <200 mg/dL, and LDL cholesterol <160 mg/dL; the results were compared with those of 50 normolipidemic (HDL cholesterol >40 mg/dL) male subjects. CETP activities were assayed in vitro and expressed as the percent of [
3
H]cholesteryl ester transferred from HDL
3
to LDL during a 16-hour incubation. In addition, postheparin plasma activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were determined in 71 patients with a low HDL cholesterol level. Distributions of CETP activities were unimodal in control subjects (mean±SD, 23.1±5.0%), but they were bimodal in the low-HDL patients. Among the latter, 27 patients had elevated CETP activities (40.8±4.6%), whereas 82 patients had CETP activities that overlapped the normal range (26.14±7.6%). Low-HDL patients with normal CETP activities had 20% lower LPL activities (
P
=.01), 25% higher HTGL activities (
P
=.03), and 63% lower LPL/HTGL ratios (
P
<.001) than those of low-HDL patients with increased CETP activity. Furthermore, mean LPL and HTGL activities in the low-HDL patients with elevated CETP activities were in the normal range. Another important distinction between the two subgroups with low HDL was that the subgroup with high CETP activity had only a 30% prevalence of coronary heart disease compared with a 70% prevalence in the subgroup with normal CETP activity (
P
<.01). These findings suggest that elevated CETP activity may be a significant factor in causing low HDL cholesterol levels in a distinct subgroup of normolipidemic patients with low HDL cholesterol levels.
Effects of Platelet-Derived Growth Factor on the Synthesis of Lipoprotein Lipase in Human Monocyte–Derived Macrophages
Authors: Inaba Toshimori , Kawamura Masako , Gotoda Takanari , Harada Kenji , Shimada Masako , Ohsuga Jun-ichi , Shimano Hitoshi , Akanuma Yasuo , Yazaki Yoshio , Yamada Nobuhiro
Published: June 2011

AbstractLipoprotein lipase (LPL), which is secreted by the two predominant cell types in atherosclerotic plaque, macrophages and smooth muscle cells, may be involved in atherosclerosis by generating atherogenic remnant lipoproteins. We investigated the effects of platelet-derived growth factor (PDGF)–BB on the synthesis of LPL by human monocyte–derived macrophages. These cells were cultured in the presence of PDGF-BB for 8 days, after which the enzyme activity, mass, and mRNA levels of LPL were determined. The effect of PDGF-BB was time-dependent and dose-dependent at concentrations of 1 to 10 ng/mL. At 10 ng/mL PDGF-BB enhanced twofold to 2.3-fold the secretion of LPL, and a pulse-labeling study with [35S]methionine revealed that 10 ng/mL PDGF-BB significantly increased the synthesis of LPL. Northern blotting analysis showed that the LPL mRNA level increased dose dependently in macrophages treated with PDGF-BB, and 10 ng/mL PDGF-BB enhanced twofold the expression of LPL mRNA. The protein kinase C inhibitor staurosporine suppressed the effect of PDGF-BB on LPL activity. These results indicate that PDGF-BB stimulated transcription of the LPL gene in human monocyte–derived macrophages through protein kinase C activation and resulted in an increased synthesis of LPL. Therefore, we hypothesize that the augmented synthesis of LPL by PDGF-BB modulates atherosclerosis by influencing lipoprotein metabolism in the vascular wall.
Early Change of Vascular Permeability in Hypercholesterolemic Rabbits
Authors: Wu Chau-Chung , Chang Shu-Wen , Chen Muh-Shy , Lee Yuan-Teh
Published: June 2011

Abstract
To study the effect of hyperlipidemia on vascular permeability, serial anterior chamber fluorophotometric examinations were carried out on 33 control rabbits (group 1) and 32 diet-induced hypercholesterolemic (group 2) rabbits. Changes in the blood-aqueous barrier function associated with total serum cholesterol (CHO) and triglyceride (TG) levels were studied at the beginning of the study and every 2 weeks thereafter for up to 16 weeks following 0.5% cholesterol–enriched diet feeding. Concurrently, a slit-lamp biomicroscope was used to examine the iris for evidence of atheromatous plaque. In group 1, the CHO level decreased slightly during the first 6 weeks and remained rather steady thereafter. The status of the blood-aqueous barrier correlated significantly with serum CHO and TG levels (
r
=.46,
P
<.001;
r
=.23,
P
=.01, respectively). In group 2, CHO and TG levels increased significantly after 2 and 8 weeks of cholesterol-enriched diet feeding, respectively. The blood-aqueous barrier also became more permeable than that in group 1 after 2 weeks’ and increased above its baseline level after 6 weeks’ feeding. Both CHO and TG levels correlated well with the degree of blood-aqueous barrier breakdown (
r
=.51,
P
<.001;
r
=.25,
P
<.001, respectively). The first evidence of iridic lipid-streak deposition was noted at 7.6±0.7 weeks, while definite iridic atheromatous plaque appeared 11.2±0.7 weeks after feeding. The change in the blood-aqueous barrier also correlated well with the semiquantitative score of iridic plaque (
r
=.58,
P
<.001) and usually preceded visual evidence of plaque formation. Pathological examinations of the eye showed marked foam cell infiltration in the stroma of the ciliary body, the ciliary process, and iris stroma, similar to changes in atheromas of the thoracic aorta after 16 weeks of feeding. In conclusion, anterior segment fluorophotometry is a valuable tool for detecting vascular integrity in vivo. These results suggest that the permeability change correlates well with serum CHO level and occurs in the very early stages of atherogenesis.
Docosahexaenoic Acid Selectively Attenuates Induction of Vascular Cell Adhesion Molecule–1 and Subsequent Monocytic Cell Adhesion to Human Endothelial Cells Stimulated by Tumor Necrosis Factor–α
Authors: Weber Christian , Erl Wolfgang , Pietsch Angelika , Danesch Ulrich , Weber Peter C.
Published: June 2011

AbstractIncorporation of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) but not eicosapentaenoic acid or n-6 arachidonic acid into human umbilical vein endothelial cell (HUVEC) phospholipids dose-dependently reduced tumor necrosis factor–α (TNF-α)–induced surface expression of vascular cell adhesion molecule–1 (VCAM-1). In parallel, DHA inhibited TNF-α–stimulated monocytic U937 cell adhesion to HUVECs but did not affect TNF-α– or interferon gamma–induced expression of intercellular adhesion molecule–1 and endothelial leukocyte adhesion molecule–1 or VCAM-1 induction by interleukin-1β. DHA appeared to attenuate VCAM-1 transcription, as it reduced induction of VCAM-1 mRNA by TNF-α. VCAM-1 induction is regulated by activation of nuclear factor–kB, which can be mediated by a TNF-α–responsive phosphatidylcholine-specific phospholipase C (PC-PLC). Gel-shift analysis showed inhibition of TNF-α–induced nuclear factor–kB mobilization by DHA. While the PC-PLC inhibitor D609 dose-dependently prevented VCAM-1 induction by TNF-α, 1,2-diacyl-glycerol (DAG) stimulated VCAM-1 expression, suggesting that VCAM-1 induction by TNF-α may be mediated by activation of PC-PLC. Treatment with DHA resulted in a fourfold enrichment in PC. In addition, DHA or D609 but not eicosapentaenoic acid or arachidonic acid suppressed activation of PC-PLC by TNF-α, estimated as [14C]DAG synthesis in prelabeled HUVECs. Incorporation of DHA into phospholipids selectively attenuates VCAM-1 induction by TNF-α and subsequent monocytic cell adhesion by inhibition of TNF-α–stimulated PC-PLC activation in HUVECs.
Megakaryocytes From Patients With Coronary Atherosclerosis Express the Inducible Nitric Oxide Synthase
Authors: de Belder A. , Radomski M. , Hancock V. , Brown A. , Moncada S. , Martin J.
Published: June 2011

Abstract
Endothelial and platelet generation of nitric oxide (NO) plays an important role in the regulation of hemostasis. Alterations in NO biosynthesis are described in atherosclerosis. We have investigated the NO pathway in megakaryocytes and platelets from patients with atherosclerosis and age-matched control subjects. Megakaryocytes and platelets were isolated from patients with severe coronary atherosclerosis (n=19) and normal coronary arteries (n=9) as demonstrated by selective angiography. Constitutive (Ca
2+
-dependent) and inducible (Ca
2+
-independent) NO synthase (cNOS and iNOS, respectively) activities were measured by using the citrulline assay and by immunostaining techniques using an anti-peptide antibody to iNOS. Megakaryocytes from patients with atherosclerosis expressed significantly greater amounts of iNOS (1.28±0.46 pmol citrulline · mg
−1
· min
−1
) than cNOS (0.29±0.40 pmol · mg
−1
· min
−1
). In contrast, megakaryocytes from patients with normal coronary arteries expressed significantly more cNOS (1.48±0.23 pmol · mg
−1
· min
−1
) than iNOS (0.49±0.40 pmol · mg
−1
· min
−1
). Platelets isolated from both groups showed no significant difference in cNOS expression, and no iNOS was seen in either group. Immunostaining confirmed the presence of the iNOS in megakaryocytes. These results suggest there is a link between the expression of iNOS in the megakaryocyte and atherosclerosis.
Factor V Leiden Gene Mutation and Thrombin Generation in Relation to the Development of Acute Stroke
Authors: Catto Andrew , Carter Angela , Ireland Helen , Bayston Trevor A. , Philippou Helen , Barrett Jennifer , Lane David A. , Grant Peter J.
Published: June 2011

Abstract
To determine the prevalence of the factor V Leiden gene mutation in relation to the phenotypes of cerebral infarction and cerebral hemorrhage, we studied 386 randomly selected cases of acute stroke and 247 control subjects. Factor V genotype was determined by amplification of a 267-bp sequence of exon/intron 10 of the factor V gene. Levels of prothrombin fragment F
1+2
, a marker of thrombin generation, were determined in both acute and convalescent stroke and related to factor V genotype. Prothrombin fragment F
1+2
was assessed by using an enzyme-linked immunosorbent assay. Sixteen stroke cases (4.1%) were identified as having the mutation compared with 14 (5.6%) control subjects. Prothrombin fragment F
1+2
levels were estimated in 191 cases and found to be elevated both acutely and after 3 months, but they were not related to factor V genotype. Prothrombin fragment F
1+2
is elevated in acute stroke and requires further evaluation in relation to cerebrovascular disease. These results suggest that the factor V Leiden gene mutation is not a risk factor for arterial thrombosis causing stroke.
Hormonal Regulation of Serum Lipoprotein(a) Levels
Authors: Olivecrona Hans , Johansson Anna G. , Lindh Erik , Ljunghall Sverker , Berglund Lars , Angelin Bo
Published: June 2011

Abstract
In response to treatment with growth hormone, serum levels of lipoprotein(a) increase, while those of LDL cholesterol decrease. To establish if increased levels of insulin-like growth factor–I may be of importance for these changes, we analyzed serum lipoprotein concentrations in 11 male patients with idiopathic osteoporosis who were treated with growth hormone (2 IU · m
−2
· d
−1
) or insulin-like growth factor–I (80 μg · kg
−1
· d
−1
) in a randomized, double-blind, crossover study. LDL cholesterol was reduced by 0.7 mmol/L (
P
<.01) during growth hormone treatment but was not affected when the same patients received insulin-like growth factor–I. In contrast, mean lipoprotein(a) levels increased from 519 to 571 mg/L (
P
<.03) in response to growth hormone but were reduced from 538 to 478 mg/L (
P
<.04) during treatment with insulin-like growth factor–I. These results indicate that growth hormone exerts its effects on lipoprotein metabolism independent of insulin-like growth factor–I. Furthermore, the results suggest that treatment with insulin-like growth factor–I may reduce lipoprotein(a) levels.
Effect of Cholesterol Lowering on Intravascular Pools of TFPI and Its Anticoagulant Potential in Type II Hyperlipoproteinemia
Authors: Hansen John-Bjarne , Huseby Kirsten Raanaas , Huseby Nils-Erik , Sandset Per Morten , Hanssen Tor-Arne , Nordøy Arne
Published: June 2011

Abstract
Tissue factor pathway inhibitor (TFPI) inhibits the extrinsic coagulation system. A major pool of TFPI is associated with the vascular endothelium and can be mobilized into the circulation by heparin. In circulating blood, TFPI is mainly associated with LDL (80%), whereas 10% to 20% is carrier free. In this study, heparin administration caused a 2.2-fold and a 7.5-fold increase in TFPI activity and TFPI antigen, respectively, in 25 patients with phenotypes IIa and IIb hyperbetalipoproteinemia. Because the antigen determination of TFPI almost exclusively measures carrier-free TFPI, more than 90% of the heparin-induced increase in TFPI activity was caused by mobilization of carrier-free TFPI from the vascular endothelium. Therapeutic lowering of total cholesterol (a decrease of 31.1±11.6%,
P
<.001) by 40 mg/d lovastatin in 17 patients with hyperbetalipoproteinemia was accompanied by a parallel decrease in TFPI activity (of 27.7±24.2%,
P
<.001) because of a reduction in LDL-TFPI complexes. However, drug intervention did not affect carrier-free TFPI or the magnitude of the vascular pool of TFPI that could be mobilized into the circulation by heparin. Moreover, this reduction of LDL-TFPI complexes did not reduce the anticoagulant potency of TFPI in plasma or of the vascular endothelial pool. The results of this study may imply that the anticoagulant potency of TFPI is associated with its carrier-free form in plasma or on the endothelium and that downregulation of LDL affects neither the size nor the anticoagulant potency of the endothelial pool of TFPI.
α-Thrombin Stimulates Urokinase Production and DNA Synthesis in Cultured Human Cerebral Microvascular Endothelial Cells
Authors: Shatos Marie A. , Orfeo Thomas , Doherty Jacqueline M. , Penar Paul L. , Collen Desire , Mann Kenneth G.
Published: June 2011

Abstract
α-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by α-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, α-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L α-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of α-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that α-thrombin may regulate repair of the cerebral microvascular system.
The Biologic Behavior of Balloon Hyperinflation–Induced Arterial Lesions in Hypercholesterolemic Pigs Depends on the Presence of Foam Cells
Authors: Recchia Dino , Abendschein Dana R. , Saffitz Jeffrey E. , Wickline Samuel A.
Published: June 2011

AbstractLack of a large-animal model of accelerated atherosclerosis has limited study of the biologic behavior of atherosclerotic lesions. We hypothesized that mechanical vascular trauma combined with diet-induced hypercholesterolemia would result in rapid development of complex atherosclerosis-like lesions. Accordingly, we induced deep injury to a carotid artery by repetitive balloon hyperinflations in minipigs that were fed either an atherogenic diet (n=30) or a standard diet (controls, n=4) and examined the resultant lesions 1 month later. The neointimal lesions that evolved in 23 patent vessels from cholesterol-fed animals were complex, exhibiting infiltration of smooth muscle and foam cells and evidence of organized thrombus, recent thrombus, hemorrhage, and calcification. Lesions were separable histologically into two groups: foam-cell rich (n=12), with 33±10 foam cells per high-power field, and foam-cell poor (n=11), with 4±1 foam cells per high-power field. Minipigs with foam cell–rich lesions had higher serum cholesterol levels than those with foam cell–poor lesions (712±178 vs 468±240 mg/dL,P<.02). The incidence of intralesional thrombus was also significantly greater in foam cell–rich than in foam cell–poor lesions (50% vs 9%,P<.04). In addition, the degree of luminal stenosis was greater in the presence of lesions containing thrombus compared with those without thrombus (60±38% vs 30±29%,P=.05). Lesions in the control animals were fibrocellular and lacked foam cells and thrombus. Thus, hypercholesterolemia appeared to affect lesion composition and behavior. Lesions with an abundance of foam cells were more likely to show evidence of intralesional thrombosis, which was associated with increased luminal stenosis. Our findings suggest that foam cells may predispose to lesion instability and thrombosis, leading to even more severe luminal obstruction.
Independent Mutations at Codon 3500 of the Apolipoprotein B Gene Are Associated With Hyperlipidemia
Authors: Gaffney Dairena , Reid Jacqueline M. , Cameron Isobel M. , Vass Keith , Caslake Muriel J. , Shepherd James , Packard Christopher J.
Published: June 2011

Abstract
The apoB arginine-to-glutamine change at codon 3500 has become established as a cause of failure of binding of the LDL particle to its receptor and the consequent hypercholesterolemia of familial defective apoB 100. A search for further similar mutations was undertaken by systematic screening of a candidate region of the apoB gene from individuals with hypercholesterolemia. Polymerase chain reaction and denaturing gradient gel electrophoresis were used. We describe two families in which a different mutation in the codon 3500 causes an arginine-to-tryptophan substitution. Most adults in these families who have this mutation have hypercholesterolemia. LDL derived from all who have inherited the mutation is dysfunctional in that it allows only poor growth of an LDL cholesterol–dependent cell line. We conclude that this arginine 3500 is essential to the function of apoB and that its loss and replacement by glutamine or tryptophan is responsible for the hypercholesterolemia of familial defective apoB 100.
Lipoprotein Lipase Mass and Activity in Plasma and Their Increase After Heparin Are Separate Parameters With Different Relations to Plasma Lipoproteins
Authors: Tornvall Per , Olivecrona Gunilla , Karpe Fredrik , Hamsten Anders , Olivecrona Thomas
Published: June 2011

AbstractLipoprotein lipase (LPL) activity and mass in plasma and their increase after heparin administration were measured in 61 men who had suffered myocardial infarction before the age of 45 years and in 69 population-based age- and sex-matched control subjects without coronary heart disease to study the relations between these parameters in plasma and their correlations with plasma lipoproteins in subjects with a wide range of lipoprotein and LPL levels. There was a relatively large amount of LPL protein compared with LPL activity in preheparin plasma, indicating that the majority of circulating LPL is catalytically inactive. LPL mass and activity in postheparin plasma (postheparin minus preheparin values) were highly correlated, and the calculated mean specific activity (0.35 mU/ng) was in the range expected for catalytically active LPL. Hence, heparin releases mainly active LPL. The four LPL parameters (mass and activity in plasma and their increase after heparin administration) were not related to each other, except for postheparin plasma LPL mass and activity, and they showed different correlations with plasma lipoprotein lipid concentrations. There was a strong positive correlation between LPL mass in preheparin plasma and the HDL cholesterol level as well as weak negative relations to VLDL triglyceride and cholesterol concentrations in the patients. In contrast, preheparin LPL activity showed no correlation with the HDL cholesterol level but weak positive relations to VLDL triglyceride and cholesterol concentrations in the control subjects. Postheparin plasma LPL activity related positively to the HDL cholesterol level and negatively to the VLDL triglyceride concentration in the control subjects. Case subjects differed from control subjects in that they had higher preheparin plasma LPL activity and a tendency toward lower specific activity of postheparin plasma LPL. The different relations of the measured LPL parameters to plasma lipoproteins and the difference in preheparin plasma LPL activity between patients and control subjects might reflect a disturbance of the LPL system in the patients.
Activation of Tissue Factor–Induced Coagulation and Endothelial Cell Dysfunction in Non–Insulin-Dependent Diabetic Patients With Microalbuminuria
Authors: Kario Kazuomi , Matsuo Takefumi , Kobayashi Hiroko , Matsuo Miyako , Sakata Toshiyuki , Miyata Toshiyuki
Published: June 2011

Abstract
We studied the relationships between albuminuria, tissue factor–induced coagulation, and endothelial cell dysfunction in 67 patients with non–insulin-dependent diabetes mellitus (NIDDM) who were divided into three groups on the basis of their urinary albumin excretion rate (AER). To assess the early phase of tissue factor–induced coagulation, activated factor VII (FVIIa) levels in plasma were measured by a direct fluorogenic assay. As markers of endothelial cell dysfunction, levels of von Willebrand factor (vWF), tissue-type plasminogen activator–plasminogen activator inhibitor–1 (TPA–PAI-1) complex, PAI-1, and tissue factor pathway inhibitor (TFPI) were measured. FVIIa levels were increased in normoalbuminuric NIDDM patients (AER <15 μg/min) when compared with normal control subjects. This FVIIa increase was accompanied by an increase in thrombin–antithrombin III complex (TAT) levels, indicating increased activation of coagulation even in normoalbuminuric patients. In NIDDM patients with microalbuminuria (AER=15-200 μg/min), the FVIIa level, the FVIIa–FVII antigen (Ag) ratio (an indicator of activation of FVII zymogen to FVIIa), and the TAT level were further increased. This group also had higher levels of endothelial cell–derived factors (vWF, TPA–PAI-1 complex, and PAI-1) than the control group. The levels of endothelial cell–derived factors (including TFPI) were highest in the NIDDM patients with overt albuminuria (AER>200 μg/min). In all 67 diabetic patients, AER showed a strong positive correlation with FVIIa (
r
=.574,
P
<.0001) and a weakly but still significant correlation with FVIIa-FVII:Ag (
r
=.365,
P
=.01), vWF (
r
=.315,
P
<.01), and TAT (
r
=.323,
P
<.01). FVIIa showed a weaker correlation with vWF (
r
=.244,
P
<.05). FVIIa generation concomitant with an increase in AER is probably due to endothelial cell damage. Increased plasma FVIIa levels would produce hypercoagulability in NIDDM patients with microalbuminuria and thus may be a risk for cardiovascular disease.
Inflammation and Matrix Metalloproteinases in the Enlarging Abdominal Aortic Aneurysm
Authors: Freestone Tim , Turner Robert J. , Coady Andrew , Higman Dan J. , Greenhalgh Roger M. , Powell Janet T.
Published: June 2011

Abstract
The risk of rupture of an abdominal aortic aneurysm increases with aortic diameter. To obtain insight into the pathological processes associated with the vascular remodeling that accompanies aortic dilatation, we compared the histological features and the activity of matrix metalloproteinases (MMPs) in biopsies from 21 small (4.0 to 5.5 cm in diameter) and 45 larger abdominal aortic aneurysms. The histological feature most clearly associated with enlarging aneurysm diameter was a higher density of inflammatory cells in the adventitia,
P
=.018. This inflammation was nonspecific, principally macrophages and B lymphocytes. Fibrosis of the adventitia provided compensatory thickening of the aortic wall as the aneurysm diameter increased. A combination of zymography and immunoblotting identified gelatinase A (MMP-2) as the principal metallogelatinase in small aneurysms, whereas zymography indicated an increasing activity of gelatinase B (MMP-9) in large aneurysms. Homogenates prepared from both small and large aneurysms had similar total activity against gelatin or type IV collagen. However, the concentration of gelatinase A, determined by immunoassay, was highest for small aneurysms: median concentrations, 385, 244, and 166 ng/mg protein for small aneurysms, large aneurysms, and atherosclerotic aorta, respectively. Immunolocalization studies indicated that gelatinase A was concentrated along fibrous tissue of both the acellular media and the atherosclerotic plaque. The recruitment of inflammatory cells into the adventitia, with subsequent elaboration of metalloproteinases, including gelatinase B, may contribute to the rapid growth and rupture of larger aneurysms.
Immunoselective Targeting of an Anti-Thrombin Agent to the Surface of Cytokine-Activated Vascular Endothelial Cells
Authors: Kiely Jeanne-Marie , Cybulsky Myron I. , Luscinskas Francis W. , Gimbrone Michael A.
Published: June 2011

AbstractAn immunoconjugate was designed to target hirudin, a potent and specific inhibitor of thrombin, to the surface of activated endothelial cells. Hirudin was covalently cross-linked to the monoclonal antibody H18/7 that recognizes the extracellular domain of E-selectin (CD62E), an endothelium-leukocyte adhesion molecule that is expressed only on cytokine-activated endothelium. The hirudin-H18/7 immunoconjugate selectively bound to interleukin-1–activated but not to unactivated cultured human umbilical vein endothelial cells with a temporal profile similar to that of inducible cell-surface procoagulant activity. When bound to activated endothelial cells, the hirudin-H18/7 immunoconjugate significantly inhibited endogenous thrombin activity generated from coincubated human plasma and fibrin clot formation on the monolayer surface. Cellular responses that are mediated via the thrombin receptor, such as increases in cytoskeletal F-actin content, also were significantly downregulated, and monolayers were protected from thrombin-induced disruption by this treatment. The ability to selectively antagonize thrombin-dependent processes at the endothelium-blood interface may provide new insights into complex pathophysiological processes, such as thrombosis, inflammation, and atherogenesis. These studies also demonstrate the general feasibility of selective targeting of therapeutic agents to endothelial cells based on recognition of an activation-dependent surface phenotype.
Upregulation of Connexin43 Gap Junctions During Early Stages of Human Coronary Atherosclerosis
Authors: Blackburn J. P. , Peters N. S. , Yeh H.-I. , Rothery S. , Green C. R. , Severs N. J.
Published: June 2011

Abstract
Interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, but little information is available on the role of direct intercellular communication via gap junctions in this process. To investigate gap junction expression in the pathogenesis of human atherosclerosis, lesions representing different stages of the disease were obtained from coronary arteries of hearts removed from patients undergoing cardiac transplantation. Twelve hearts, each providing 1 to 3 segments of artery, were used in the study. Sections were examined by confocal laser scanning microscopy after immunofluorescent labeling with a specific antibody against connexin43, the major gap-junctional protein of smooth muscle cells, to permit high-definition visualization of immunolabeled gap junctions through the depth of the specimen. Double labeling using anti-connexin43 and cell type–specific antibodies demonstrated colocalization of gap junctions with smooth muscle cells but not with macrophages, a relationship confirmed by electron microscopy. Regions of intimal thickening and early atheromatous lesions showed markedly increased expression of connexin43 gap junctions between intimal smooth muscle cells compared with the undiseased vessel. This increase in gap junctions was most marked in regions of intimal thickening, semiquantitative analysis of the confocal digital images revealing a >10-fold increase compared with the undiseased vessel. The quantity of labeled gap junctions in early atheromatous lesions, although higher than that of the undiseased vessel, was lower than that of intimal thickenings, and this trend toward reduced levels of gap junction immunolabeling with lesion progression continued, the value observed in the most advanced atheromatous lesions being lower than that of the undiseased vessel. As the quantity of gap junctions declined, their distribution became more patchy and the sizes of individual junctions larger. The results suggest that enhanced expression of gap junctions between smooth muscle cells may play a role in maintaining the synthetic phenotype during early growth of the atherosclerotic plaque.
Higher Plasma Homocyst(e)ine and Increased Susceptibility to Adverse Effects of Low Folate in Early Familial Coronary Artery Disease
Authors: Hopkins Paul N. , Wu Lily L. , Wu James , Hunt Steven C. , James Brent C. , Vincent G. Michael , Williams Roger R.
Published: June 2011

Abstract
To examine the graded risks for coronary artery disease (CAD) associated with plasma homocyst(e)ine [H(e)] and to evaluate the extent to which this risk is mediated by altered vitamin status, we measured plasma concentrations of H(e), vitamins B
6
and B
12
, and folate as well as other coronary risk factors in subjects with early familial CAD and in control subjects. We studied 120 male and 42 female patients with early CAD who were unrelated to each other but were from families in which at least one other sibling had early CAD. Control subjects were 85 men and 70 women with the same age range (38 to 68) as the subjects with CAD at screening. Increasing H(e) was associated with graded increased risks of CAD that appeared consistent with a multiplicative model. Relative odds for CAD were approximately 12.8 in women when those with H(e) levels of 19 μmol/L and above were compared with those with H(e) levels of 9 μmol/L or less (
P
=.007). For men, the same comparison yielded relative odds of 13.8 (
P
=.0002). Plasma H(e) remained a strong, independent risk factor after adjustment for standard risk factors and plasma vitamin levels in multiple logistic regression (relative odds, 8.1 for a 10-μmol/L increase in H(e); 95% confidence interval, 3.2 to 20.4;
P
<.0001). In multivariate ANCOVA the slope of H(e) versus folate was much steeper in subjects with CAD than in control subjects (
P
=.0035). These data suggest that high plasma H(e) is an important, independent contributor to risk for early familial CAD. Furthermore, subjects with early familial CAD who had low plasma folate levels had exaggerated elevations in plasma H(e), suggesting a possible genetic sensitivity to the detrimental effects of lower folate intake.
Recombinant Leech Antiplatelet Protein Specifically Blocks Platelet Deposition on Collagen Surfaces Under Flow Conditions
Authors: van Zanten G. Henrita , Connolly Thomas M. , Schiphorst Marion E. , de Graaf Sytske , Slootweg Pieter J. , Sixma Jan J.
Published: June 2011

Abstract
Salivary glands of the leech
Haementeria officinalis
contain a protein, leech antiplatelet protein (LAPP). This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions. In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions. rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC
50
values of 70, 600, and 90 nmol/L, respectively (shear rate=1600 s
−1
). Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s
−1
. rLAPP caused a concentration-dependent inhibition of binding of
125
I–von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours. Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect. Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 μmol/L rLAPP). rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells. rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.