bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Novel Molecular Defects in theLCATGene Are Associated With Fish Eye Disease
Authors: Kuivenhoven Jan Albert , Stalenhoef Anton F.H. , Hill John S. , Demacker Pierre N.M. , Errami A. , Kastelein John J.P. , Pritchard P. Haydn
Published: June 2011

AbstractA 53-year-old man with a severely reduced HDL cholesterol level, dense corneal opacities, normal renal function, and premature coronary artery disease was investigated together with 16 members of his family. The proband was diagnosed with fish eye disease. As in previously reported patients with fish eye disease, the endogenous plasma cholesterol esterification rate was near normal, yet lecithin:cholesterol acyltransferase (LCAT) activity was almost absent when measured with exogenous HDL analogues used as substrate. Direct sequencing of theLCATgene revealed two novel missense mutations in exon 1 and exon 4, resulting in the substitution of Pro10with Gln (P10Q) and Arg135with Gln (R135Q), respectively. Both missense mutations were located on different alleles. Genetic analysis by polymerase chain reaction revealed 4 carriers of the P10Q and 3 carriers of the R135Q defect. Functional assessment of both missense mutations revealed that when exogenous HDL analogues were used as substrate, the specific activity of rLCATP10Qwas 18% of wild type (WT); however, when LDL was used as substrate, the activity was 146% of WT. By contrast, rLCATR135Qwas inactive against both substrates. Thus, we conclude that theLCATR135Dmutation is causative for complete LCAT deficiency and that the clinical phenotype of fish eye disease seen in this patient is due to the Pro10mutation. The presence of premature coronary artery disease in the absence of other risk factors in this new case of fish eye disease raises questions regarding the risk of atherosclerosis, which has previously been reported to be nonexistent.
Only the Two End Helixes of Eight Tandem Amphipathic Helical Domains of Human Apo A-I Have Significant Lipid Affinity
Authors: Palgunachari Mayakonda N. , Mishra Vinod K. , Lund-Katz Sissel , Phillips Michael C. , Adeyeye Samuel O. , Alluri Sridevi , Anantharamaiah G.M. , Segrest Jere P.
Published: June 2011

Abstract
Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic α-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the
N
- and
C
-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-
sn
-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to–liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the
N
- and
C
-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to–liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.
Antithrombin III and Fibrinogen as Predictors of Cardiac Events in Patients With Angina Pectoris
Authors: Thompson S.G. , Fechtrup C. , Squire E. , Heyse U. , Breithardt G. , van de Loo J.C.W. , Kienast J.
Published: June 2011

AbstractBecause measurements of hemostatic factors might aid the prediction of cardiovascular clinical events, we investigated the long-term prognostic importance of selected hemostatic factors in patients with angina pectoris. At recruitment, 209 patients underwent clinical assessment and coronary angiography, and a range of hemostatic factors were measured. During the follow-up period of 9 years, 58 patients (28%) suffered a cardiac event (acute myocardial infarction or death from cardiac causes). The risk of cardiac events was positively related to baseline measurements of fibrinogen (risk ratio per SD [RR] increase 1.29, 95% confidence interval [CI] 0.99 to 1.68,P=.06) and negatively related to antithrombin III activity measurements (RR 0.75, 95% CI 0.59 to 0.95,P=.02). No other hemostatic factor measured was significantly related to the risk of having a cardiac event. Worsening of angina in the few weeks before and ejection fraction evaluation at the initial angiography were both strongly related to the risk of cardiac events. However, the relationships of fibrinogen and antithrombin III measurements to risk remained almost unchanged after adjusting for worsening of angina and ejection fraction. Fibrinogen and antithrombin III may have an important etiologic role in the prognosis of patients with angina pectoris.
Lys and Fibrinogen Binding of Wild-Type (Trp72) and Mutant (Arg72) Human Apo(a) Kringle IV-10 Expressed in
E coli
and CHO Cells
Authors: Klezovitch Olga , Scanu Angelo M.
Published: June 2011

Abstract
In a previous study, we identified a lysine (Lys)-binding–defective form of human lipoprotein(a) and attributed this defect to the presence of a Trp72→Arg mutation in apolipoprotein(a) [apo(a)] kringle IV-10. To document this relationship, we expressed both wild-type (wt) and mutant (mut) forms of kringle IV-10 in
Escherichia coli
(nonglycosylated form) and Chinese hamster ovary (CHO) cells (glycosylated form). The Arg72 mut was prepared by introducing the T→A mutation in apo(a) kringle IV-10 amplified from human liver mRNA by the reverse-transcriptase polymerase chain reaction technique. All expressed kringles were tested for their ability to bind Lys and plasmin-modified fibrinogen (PM-fibrinogen). wt kringle IV-10 expressed in both
E coli
and CHO cells bound to Lys-Sepharose with comparable affinity. In contrast, the Arg72 mut expressed in both systems exhibited no Lys-binding capacity. Moreover, the wt kringle IV-10 expressed in both systems bound to PM-fibrinogen and exhibited two binding components, one Lys mediated (inhibitable by ε-amino-
n
-caproic acid) and one Lys insensitive, occurring in about the same proportions. Only the latter type of binding was present in the Arg72 mut expressed in
E coli.
We conclude that kringle IV-10 of human apo(a) has Lys-and PM-fibrinogen–binding capacities that are independent of glycosylation and require the presence of Trp72, one of the seven amino acids that constitute the Lys-binding site of kringle IV-10. Our results also show that the binding of kringle IV-10 to PM-fibrinogen is more complex than that to Lys, in that the former requires an additional binding site or sites outside the Lys-binding site.
Evidence of Impaired Glomerular Charge Selectivity in Nondiabetic Subjects With Microalbuminuria
Authors: Kofoed-Enevoldsen Allan , Foyle Wendy-Jane , Fernandez Maryam , Yudkin John S.
Published: June 2011

Abstract
Microalbuminuria is associated with excess cardiovascular morbidity and mortality in diabetic and nondiabetic subjects. Loss of glomerular charge selectivity may explain the development of microalbuminuria in diabetic subjects. The primary population in this cross-sectional study was 124 subjects aged 40 to 75 years without glucose intolerance and with a previous (3 years before the present study) urinary albumin excretion rate (UAE) in the normal (<20 μg/min) or microalbuminuric (>20 μg/min) range. The secondary population consisted of 39 offspring aged 15 to 40 years. The main outcome measures included UAE, urinary IgG/IgG4 selectivity index (SI), and the presence of ischemic heart disease as determined by questionnaire or ECG. Among the primary population, a significant inverse correlation was found between SI and UAE (
r
=−.40,
P
<.001). Reduction in SI could be demonstrated in subjects with UAE >10 μg/min. In multiple regression analysis reduction in SI was found with increasing age, independent of UAE. In 20 subjects with clinical cardiovascular disease a reduction in SI was found (1.9±0.6 versus 2.6±1.3,
P
=.001) without concomitant elevation in UAE (
P
=.99). Offspring from parents with microalbuminuria had an SI comparable to offspring from parents with normal UAE (2.7±0.7 versus 3.3±1.6,
P
=.17). In nondiabetic subjects the development of microalbuminuria is associated with reduced SI, suggesting impairment of glomerular charge selectivity. SI may offer a more sensitive monitoring of abnormalities in glomerular permselectivity than does measurement of UAE, but the ability of SI to predict development of cardiovascular disease needs to be evaluated prospectively.
Factor VII Hyperactivity and Endothelial Cell Damage Are Found in Elderly Hypertensives Only When Concomitant With Microalbuminuria
Authors: Kario Kazuomi , Matsuo Takefumi , Kobayashi Hiroko , Matsuo Miyako , Sakata Toshiyuki , Miyata Toshiyuki , Shimada Kazuyuki
Published: June 2011

Abstract
We studied the relationships among albuminuria, factor VII (FVII) hyperactivity, and endothelial cell damage in 61 elderly hypertensive subjects. The plasma levels of activated FVII (FVIIa), FVII coagulant activity, FVII antigen (FVIIag), von Willebrand factor (vWF), and thrombomodulin were measured to assess FVII hyperactivity and endothelial cell damage, and urinary albumin excretion rate (UAE) was calculated using 12-hour nighttime (7
pm
to 7
am
) urine collection (mean for 2 consecutive nights). We performed 24-hour ambulatory blood pressure monitoring in all 61 hypertensive patients and classified them into a white-coat hypertension group (n=12) and a sustained hypertension group (n=49). For the levels of FVII, vWF, and thrombomodulin, there were no differences between the white-coat hypertension group and normotensive control subjects (n=25). In the sustained hypertensive group, only the microalbuminuric subgroup (UAE, 15 to 300 μg/min: n=30) showed significant elevation compared with the normotensive group for the level of FVIIa (mean [95% confidence interval]: 4.0 [3.6 to 4.4] versus 3.0 [2.6 to 3.3] ng/mL,
P
<.001), the FVIIa/FVIIag ratio (an indicator of activation of FVII zymogen to FVIIa) (1.33 [1.19 to 1.50] versus 1.04 [0.92 to 1.19],
P
<.01), the level of vWF (188 [165 to 214] % versus 144 [129 to 160] %,
P
<.01), and thrombomodulin (11.7 [10.3 to 13.3] versus 9.3 [8.5 to 10.3] ng/mL,
P
<.01). In contrast, none of these levels in the normoalbuminuric hypertensive group (UAE <15 μg/min, n=19) differed from that in the normotensive control group. These results suggest that among elderly hypertensives, only those with microalbuminuria show enhancement of FVII activation and endothelial cell damage, while patients with white-coat hypertension and normoalbuminuric hypertensives do not show these accompanying abnormalities. Thus, increased levels of FVII activity and markers of endothelial cell damage might account for the higher risk of cardiovascular events in essential hypertension with microalbuminuria.
Gemfibrozil Treatment of Combined Hyperlipoproteinemia
Authors: Bröijersen Anders , Eriksson Mats , Wiman Björn , Angelin Bo , Hjemdahl Paul
Published: June 2011

Abstract
Hypertriglyceridemia is linked to impaired fibrinolytic function, and lipid-lowering treatment with fibric acid derivatives could hypothetically improve fibrinolysis in this condition. We therefore conducted a double-blind, placebo-controlled, crossover study of gemfibrozil treatment on fibrinolytic function in 21 men with combined hyperlipoproteinemia. Measurements were performed at rest and during mental stress and after venous occlusion. The patients had clearly disturbed fibrinolytic function, with elevated plasminogen activator inhibitor–1 (PAI-1) activity at rest (≈25 U/mL; reference, <15 U/mL). Gemfibrozil reduced plasma total and VLDL cholesterol as well as all triglyceride fractions, whereas HDL cholesterol increased (
P
<.001 for all). Total triglyceride levels were reduced by 57±4% (from 5.3 to 2.1 mmol/L). Fasting serum insulin levels were not altered by gemfibrozil treatment. Plasma levels of PAI-1 activity and tissue-type plasminogen activator (TPA) activity or antigen were unaffected by gemfibrozil treatment both at rest and during the provocations. The levels of
d-
dimer, plasmin/antiplasmin complex, and fibrinogen were also uninfluenced by gemfibrozil treatment. Mental stress elevated plasma TPA (
P
=.0036) and lowered PAI-1 (
P
=.0012) activity during placebo but not gemfibrozil treatment (
P
=.28 and
P
=.17, respectively), but treatment effects did not differ by ANOVA on Δ values (ie, stress minus rest). Venous occlusion reduced PAI-1 activity, whereas TPA and plasmin/antiplasmin complex increased during both treatments. Thus, gemfibrozil treatment did not improve fibrinolysis or lower fibrinogen levels in men with combined hyperlipoproteinemia despite marked reduction of plasma triglyceride levels. It seems unlikely that improved fibrinolysis explains the primary preventive effect of gemfibrozil.
Apolipoproteins B, (a), and E Accumulate in the Morphologically Early Lesion of ‘Degenerative’ Valvular Aortic Stenosis
Authors: O’Brien Kevin D. , Reichenbach Dennis D. , Marcovina Santica M. , Kuusisto Johanna , Alpers Charles E. , Otto Catherine M.
Published: June 2011

Abstract
Nonrheumatic aortic stenosis of trileaflet aortic valves has been considered to be a “degenerative” process, but the early lesion of aortic stenosis contains the chronic inflammatory cells, macrophages and T lymphocytes. Because lipoprotein deposition is prominent in atherosclerosis, another chronic inflammatory process, this study examined whether lipoproteins accumulate in aortic valve lesions. Immunohistochemical studies were performed to detect apolipoprotein (apo) B, apo(a), apoE, macrophages, and α-actin–expressing cells on 18 trileaflet aortic valves that ranged from normal to stenotic. All three apolipoproteins were detected in early through end-stage lesions of aortic stenosis but not in histologically normal regions. Comparison with oil red O staining suggested that most of the extracellular neutral lipid in these valves was associated with either plasma-derived or locally produced apolipoproteins. Thus, in early through end-stage aortic valve lesions, apolipoproteins accumulate and are associated with the majority of extracellular valve lipid. These results are consistent with the hypothesis that lipoprotein accumulation in the aortic valve contributes to pathogenesis of aortic stenosis.
Lipoprotein Subclasses in the Monitored Atherosclerosis Regression Study (MARS)
Authors: Mack Wendy J. , Krauss Ronald M. , Hodis Howard N.
Published: June 2011

Abstract
Accumulating evidence suggests that triglyceride-rich lipoproteins contribute to coronary artery disease. Using data from the Monitored Atherosclerosis Regression Study, an angiographic trial of middle-aged men and women randomized to lovastatin or placebo, we investigated relationships between lipoprotein subclasses and progression of coronary artery atherosclerosis. Coronary artery lesion progression was determined by quantitative coronary angiography in low-grade (<50% diameter stenosis), high-grade (≥50% diameter stenosis), and all coronary artery lesions in 220 baseline/2-year angiogram pairs. Analytical ultracentrifugation was used to measure lipoprotein masses that were statistically evaluated for treatment group differences and relationships to progression of coronary artery atherosclerosis. All low density lipoprotein (LDL), intermediate density lipoprotein (IDL), and very low density lipoprotein (VLDL) masses were significantly lowered and all high density lipoprotein (HDL) masses were significantly raised with lovastatin therapy. The mass of smallest LDL (Svedberg flotation rate [S
f
] 0 to 3), IDL (S
f
12 to 20), all VLDL subclasses (S
f
20 to 60, S
f
60 to 100, and S
f
100 to 400), and peak LDL flotation rate were significantly related to the progression of coronary artery lesions, specifically low-grade lesions. Greater baseline levels of HDL
3
were related to a lower likelihood of coronary artery lesion progression. In multivariate analyses, small VLDL (S
f
20 to 60) and HDL
3
mass were the most important correlates of coronary artery lesion progression. These results provide further evidence for the importance of triglyceride-rich lipoproteins in the progression of coronary artery disease. In addition, these results present new evidence for the possible protective role of HDL
3
in the progression of coronary artery lesions. More specific information on coronary artery lesion progression may be obtained through the study of specific apolipoprotein B–containing lipoproteins.
The Yin and Yang of Oxidation in the Development of the Fatty Streak
Authors: Navab Mohamad , Berliner Judith A. , Watson Andrew D. , Hama Susan Y. , Territo Mary C. , Lusis Aldons J. , Shih Diana M. , Van Lenten Brian J. , Frank Joy S. , Demer Linda L. , Edwards Peter A. , Fogelman Alan M.
Published: June 2011

Recent data support the hypothesis that the fatty streak develops in response to specific phospholipids contained in LDL that become trapped in the artery wall and become oxidized as a result of exposure to the oxidative waste of the artery wall cells. The antioxidants present within both LDL and the microenvironments in which LDL is trapped function to prevent the formation of these biologically active, oxidized lipids. Enzymes associated with LDL and HDL (eg, platelet activating factor acetylhydrolase) or with HDL alone (eg, paraoxonase) destroy these biologically active lipids. The regulation and expression of these enzymes are determined genetically and are also significantly modified by environmental influences, including the acute-phase response or an atherogenic diet. The balance of these multiple factors leads to an induction or suppression of the inflammatory response in the artery wall and determines the clinical course.
Probucol Downregulates E-Selectin Expression on Cultured Human Vascular Endothelial Cells
Authors: Kaneko Masayo , Hayashi Joichiro , Saito Ichiro , Miyasaka Nobuyuki
Published: June 2011

Probucol, which inhibits monocyte adhesion, is a potent antioxidant to vascular endothelium in the cholesterol-fed rabbit. The accumulation of macrophages in the lesion is influenced by increased expression of specific adhesion molecules on vascular endothelial cells. We investigated the effect of probucol on the expression of cell adhesion molecules in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were treated with lipopolysaccharide in the presence or absence of probucol (0 to 5 μmol/L) and assayed for the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin by cell–enzyme-linked immunosorbent assay. Probucol significantly downregulated the expression of E-selectin on HUVECs in a dose-dependent manner. In contrast, the expression of ICAM-1 was not affected. E-selectin but not ICAM-1 mRNA expression on HUVECs was also significantly inhibited by probucol in a dose-dependent manner. We also examined whether probucol affects cellular binding between the human monocytic cell line U937 and lipopolysaccharide-stimulated HUVECs by using an in vitro binding assay and found that probucol significantly suppressed their mutual binding in a dose-dependent manner. These data indicate a novel mechanism of action for probucol to reduce the development of atherosclerotic lesions in hyperlipidemic states.
Evidence for a Major Gene Elevating Serum Bilirubin Concentration in Utah Pedigrees
Authors: Hunt Steven C. , Wu Lily L. , Hopkins Paul N. , Williams Roger R.
Published: June 2011

In case-control studies, lower serum bilirubin levels have been associated with increased risk of developing coronary heart disease (CHD). We have also previously shown that serum bilirubin has a significant polygenic component. The purpose of the present investigation was to determine whether there was statistical evidence for a major gene explaining a significant portion of individual variation in serum total bilirubin levels and whether this gene might alter the risk of CHD. Serum bilirubin measurements were obtained from 1240 adults in 84 Utah pedigrees screened twice: from 1980 to 1983 and again from 1983 to 1986. Bivariate maximum-likelihood segregation analysis of serum bilirubin levels obtained from the two clinic visits indicated that a major gene was responsible for elevated levels in 11.5% of the persons in these pedigrees. Phenotypic variations in visit 1 bilirubin arising from polygenes were highly correlated with the phenotypic variation due to polygenes in visit 2 bilirubin, indicating a stable genetic contribution to bilirubin over 2.5 years of follow-up. The major gene explained 27% and 28% of the variance in bilirubin levels at visit 1 and visit 2, respectively. There were no correlations of unmeasured environmental factors influencing bilirubin between the two clinic visits. At both visits, persons with early CHD had lower levels of bilirubin than unaffected persons (
P
<.01). The odds ratio for the risk of CHD in the high-homozygote group was 0.31,
P
=.09. We conclude that there is a major gene modestly raising serum bilirubin levels. Since cross-sectional data indicate that low serum bilirubin levels increase the risk of CHD, this major gene may be protective against CHD for about 12% of the persons in this study.
Quantitative Assessment of Aortic Atherosclerosis in APOE*3 Leiden Transgenic Mice and Its Relationship to Serum Cholesterol Exposure
Authors: Groot Pieter H.E. , van Vlijmen Bart J.M. , Benson G. Martin , Hofker Marten H. , Schiffelers Raymond , Vidgeon-Hart Martin , Havekes Louis M.
Published: June 2011

Transgenic mice overexpressing the human dysfunctional apolipoprotein E variant, APOE*3 Leiden, develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. In the present study, we investigated the effects of diet composition and feeding period on serum cholesterol exposure and the amount of atherosclerosis in the aortic sinus in these mice, using quantitative image analysis. On each of the three diets tested—a low-fat diet, a high-saturated-fat/cholesterol diet, and a high saturated-fat/high-cholesterol/0.5%-cholate diet—transgenic animals showed a marked hyperlipidemia compared with nontransgenic littermates. Measurement of the atherosclerotic lesion areas in cross sections of the aortic sinus in animals exposed to these three diets for up to 6 months showed a 5 to 10 times greater lesion area in transgenic mice compared with nontransgenic controls. Highly significant positive correlations were found between the log-transformed data on lesion area and serum cholesterol exposure (
r
=.82 to .85 for the 1-, 2-, and 3-month treatment groups), indicating that the hyperlipidemia is likely to be a major determinant in lesion formation. On the basis of these findings, we suggest that the APOE*3 Leiden mouse represents a promising model for intervention studies with hypolipidemic and antiatherosclerotic drugs.
Risk Factors Related to Carotid Intima-Media Thickness and Plaque in Children With Familial Hypercholesterolemia and Control Subjects
Authors: Tonstad Serena , Joakimsen Oddmund , Stensland-Bugge Eva , Leren Trond P. , Ose Leiv , Russell David , Bønaa Kaare H.
Published: June 2011

To assess the relationship between risk factors for cardiovascular disease and early atherosclerotic changes in the carotid artery, we measured carotid intima-media thickness by B-mode ultrasonography in 61 boys and 29 girls 10 to 19 years old with familial hypercholesterolemia (FH) and 30 control subjects matched for age and sex. All were nonsmokers, and all the FH adolescents had a known mutation in the LDL receptor gene. Mean intima-media thickness in the far wall of the carotid bulb was greater (
P
=.03) in the FH group than in the control subjects: 0.54 mm (95% confidence interval [CI], 0.52 to 0.56) versus 0.50 mm (95% CI, 0.47 to 0.52). In the entire group, mean and maximum intima-media thicknesses in the carotid bulb were positively associated with levels of apolipoprotein B and fibrinogen after control for pubertal stage (
r
=.19 to .24;
P
<.05), as was male sex. Plasma total homocysteine was similar in the FH and control groups and was associated with mean and maximum intima-media thicknesses in the far wall of the common carotid artery and carotid bulb after control for pubertal stage (
r
=.22 to .28;
P
<.05). With the exception of the relation between plasma fibrinogen level and mean carotid bulb intima-media thickness, these associations were essentially unchanged in stepwise multiple linear regression analyses, allowing for the entry of BMI and level of HDL cholesterol into the analysis. Carotid artery plaque was present in 10% of the children with FH versus none of the control subjects. Children with plaque had a higher mean cholesterol-years score than children without plaque. These findings suggest that the classic lipid and hemostatic risk factors as well as plasma total homocysteine are associated with markers of early carotid atherosclerosis from the second decade of life. B-mode ultrasonography may prove to be a useful tool in risk stratification of children with FH.
Monocyte-Induced Downregulation of Nitric Oxide Synthase in Cultured Aortic Endothelial Cells
Authors: Marczin Nándor , Antonov Alexander , Papapetropoulos Andreas , Munn David H. , Virmani Renu , Kolodgie Frank D. , Gerrity Ross , Catravas John D.
Published: June 2011

Since endothelium-dependent vasodilation is altered in atherosclerosis and enhanced monocyte/endothelial interactions are implicated in early atherosclerosis, we evaluated the effects of monocytes on the endothelial nitric oxide (NO) pathway by estimating release of biologically active NO from cultured endothelial cells and levels of constitutive NO synthase (ecNOS). NO release was estimated in a short-term bioassay using endothelial cell–induced cGMP accumulation in vascular smooth muscle (SM) cells. Exposure of SM cells to porcine aortic endothelial cells (PAECs) and human aortic endothelial cells (HAECs) produced large increases in SM cGMP content; this increase was prevented by
N
G
-nitro-
l
-arginine methyl ester, the inhibitor of endothelial NOS. Confluent monolayers of PAECs and HAECs cocultured with monocytes also stimulated SM cGMP formation; however, NO release from these cultures was attenuated in a coculture time (2 to 48 hours)- and monocyte concentration (20 to 200×10
3
per well)–dependent manner. This effect of monocyte adhesion appeared to be selective for NO release since other biochemical pathways, such as atriopeptin- and isoproterenol-induced cyclic nucleotide accumulation within the endothelial cells, were not altered by monocytes. The effects of adherent monocytes on NO release were mimicked by monocyte-derived cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1α. Furthermore, the conditioned medium of monocytes contained significant quantities of these cytokines. Conditioned medium, as well as monocytes physically separated from the endothelial cells, attenuated NO release, suggesting that soluble factors may mediate the effects of monocytes. An IL-1β neutralizing antibody fully prevented the NO dysfunction in response to directly adherent monocytes. Superoxide dismutase, catalase, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and exogenous
l
-arginine failed to improve NO release, suggesting that oxidant stress–induced inactivation of NO or limited substrate availability were not primarily responsible for the inhibiting effects of monocytes. Western blot analysis revealed reduced quantities of ecNOS in monocyte/endothelium cocultures, as well as in HAECs treated with monocyte-conditioned medium or TNF-α. Thus, adhesion of monocytes to endothelial cells and monocyte-derived secretory products downregulate steady state levels of ecNOS, an event associated with attenuated release of biologically active NO. This mechanism may potentially contribute to diminished endothelium-dependent and NO-mediated vasodilation in early atherosclerosis.
An Increased Uptake of Prothrombin, Antithrombin, and Fibrinogen by the Rabbit Balloon-Deendothelialized Aorta Surface In Vivo Is Maintained Until Reendothelialization Is Complete
Authors: Hatton Mark W.C. , Southward Suzanne M.R. , Ross-Ouellet Bonnie , DeReske Marnie , Blajchman Morris A. , Richardson Mary
Published: June 2011

The ability of the rabbit aorta intima-media (IM) layer to adsorb certain plasma proteins was measured for up to 20 months after a deendothelializing injury in vivo. Purified radioiodinated rabbit fibrinogen, antithrombin, or prothrombin was injected intravenously into either uninjured or sham-injured rabbits (controls) or rabbits at various times (5 minutes to 20 months) after a balloon-catheter injury to the aorta. After a 10-minute circulation time, a blood sample was taken, and the rabbit was exsanguinated rapidly (via a carotid cannula) and the aorta excised. Uptake of each radiolabeled protein was measured as bound radioactivity per square centimeter of platelet- or endothelium-free aorta IM and was compared with the radioactivity (ergo concentration) in blood at exsanguination. Fibrinogen adsorption by the IM was maximal at 5 minutes after injury (10.9±2.3 pmol/cm2IM) and declined slowly to 4 to 6 pmol/cm2at 12 months (controls: 0.8±0.1 pmol/cm2). Uptake of prothrombin (3.7±0.5 pmol/cm2at 5 minutes) decreased to ≈2 pmol/cm2at 12 months (controls: 0.3 pmol/cm2). Antithrombin adsorption by the IM (3.3±0.4 pmol/cm2at 5 minutes) paralleled that of prothrombin over 12 months (controls: 0.3 to 0.4 pmol/cm2), the molar ratio ranging from 0.8 to 1.2. At 20 months, the ballooned aorta had a significantly thickened intima and was ≈90% reendothelialized. Injection of horseradish peroxidase (HRP) into rabbits at 1 or 12 months after balloon injury showed clearly that HRP activity was present throughout the entire depth of the deendothelialized, but not the reendothelialized, thickened intima. These results may indicate that an elevated turnover of hemostatic proteins continues within the deendothelialized intima after injury, conceivably until reendothelialization is complete.
Complement C5b-9 Increases Plasminogen Binding and Activation on Human Endothelial Cells
Authors: Christiansen Victoria J. , Sims Peter J. , Hamilton Karen K.
Published: June 2011

Deposition of the terminal complement proteins (C5b-9) on human endothelial cells can result in cell lysis or nonlytic alterations of cell function including procoagulant responses. Because regulation of fibrinolysis is a central endothelial function and because C9 contains a carboxyl-terminal lysine similar to other proteins that bind and facilitate activation of plasminogen (PG), the effects of complement injury on PG binding and activation on these cells were investigated. Activation of complement through deposition of C5b67 complexes on endothelial cells resulted in a small increase (≈20%) in PG binding. Incorporation of C8 into C5b-8 resulted in no further increase in binding; however, specific125I-PG binding was increased by ≈100% after C5b-9 deposition. Moreover, PG was found to bind specifically to C7 and C9. The PG bound to endothelial cells after C5b-9 deposition was readily activated by tissue-type plasminogen activator (TPA). In a cell-free system, complement C9 and a synthetic peptide composed of the 20 carboxyl-terminal amino acids of C9 enhanced PG activation by TPA. Removal of the carboxyl-terminal lysine of C9 abolished the enhancement of PG activation without diminishing PG binding. We conclude that membrane C9 may comprise a binding site for PG and serve to enhance activation of this zymogen by TPA. These findings suggest that immune injury to the endothelium may enhance both the fibrin-generating and fibrinolytic capacity of the vessel wall.
Syndecan-4 Is a Primary-Response Gene Induced by Basic Fibroblast Growth Factor and Arterial Injury in Vascular Smooth Muscle Cells
Authors: Cizmeci-Smith Gunay , Langan Eugene , Youkey Jerry , Showalter Lori Jo , Carey David J.
Published: June 2011

Syndecans are a family of transmembrane proteoglycans that have been implicated in cell–extracellular matrix adhesion and growth factor binding. We reported previously that syndecan-1 expression by cultured rat vascular smooth muscle cells (VSMCs) is induced by serum- or platelet-derived growth factor (PDGF). We now report that syndecan-4 mRNA is rapidly induced in cultured VSMCs in response to basic fibroblast growth factor (bFGF) or serum stimulation. In the presence of cycloheximide, induction of syndecan-4 mRNA was enhanced. These characteristics identified syndecan-4 as a primary-response gene product in VSMCs. In contrast, syndecan-1 mRNA expression in response to serum was completely blocked in the presence of cycloheximide. We also examined the expression of syndecan mRNAs in VSMCs in response to balloon catheter injury in vivo. A reverse transcriptase–polymerase chain reaction technique was developed that enabled us to amplify all four syndecan mRNAs in a single reaction tube and determine relative changes in their expression. All four syndecan mRNAs were detected in uninjured rat carotid arteries. In endothelium-denuded arteries, the medial layer (presumably VSMCs) accounted for 70% to 90% of the syndecan mRNAs in the vessel wall. The levels of syndecan-2 and syndecan-3 mRNAs were not altered significantly after balloon injury. In contrast, syndecan-4 mRNA was increased at early times after injury but then decreased to control level by 7 days. Syndecan-1 mRNA levels showed a slower but prolonged increase that reached a maximum at 7 days after injury. Immunostaining with anti–syndecan-4 antibodies demonstrated a rapid increase in syndecan-4 proteoglycan expression in the injured carotid artery.
Neutralizing Antibodies Directed Against Osteopontin Inhibit Rat Carotid Neointimal Thickening After Endothelial Denudation
Authors: Liaw Lucy , Lombardi Donna M. , Almeida Manuela M. , Schwartz Stephen M. , deBlois Denis , Giachelli Cecilia M.
Published: June 2011

Osteopontin is an arginine-glycine-aspartate-containing acidic glycoprotein with adhesive and migratory activities in vitro. We previously showed that osteopontin was highly expressed in injured rat arteries as well as in human atherosclerotic plaques. In contrast, uninjured blood vessels make very little osteopontin. In this report, we have investigated the role of osteopontin in rat neointima formation using neutralizing antibodies. Rats were treated with either nonimmune or antiosteopontin antibody and subjected to endothelial denudation of the carotid artery by using a balloon catheter. Two weeks after injury, intimal areas and cell numbers were significantly decreased (33% and 31%, respectively) in the antiosteopontin group compared with the nonimmune IgG group. No differences in carotid medial areas or cell numbers were observed. Intimal and medial replication rates, as measured by continuous bromodeoxyuridine infusion during the final week of the experimental protocol, were not significantly different between the two groups. No gross histological changes were noted in the intimas formed in the presence of either neutralizing or nonimmune antibody. In addition, no difference in early carotid medial cell replication rate was observed when antibodies were infused for 4 days after angioplasty. These data demonstrate for the first time a functional role for osteopontin in the process of carotid neointimal thickening in vivo and suggest that osteopontin plays an active role in the remodeling processes important for human atherosclerotic and restenotic lesion development.
Effects of Serum Lipoproteins and Smoking on Atherosclerosis in Young Men and Women
Authors: McGill Henry C. , McMahan C. Alex , Malcom Gray T. , Oalmann Margaret C. , Strong Jack P.
Published: June 2011

Atherosclerosis begins in childhood and progresses from fatty streaks to raised lesions in adolescence and young adulthood. A cooperative multicenter study (Pathobiological Determinants of Atherosclerosis in Youth [PDAY]) examined the relation of risk factors for adult coronary heart disease to atherosclerosis in 1079 men and 364 women 15 through 34 years of age, both black and white, who died of external causes and were autopsied in forensic laboratories. We quantitated atherosclerosis of the aorta and right coronary artery as the extent of intimal surface involved by fatty streaks and raised lesions and analyzed postmortem serum for lipoprotein cholesterol and thiocyanate (as an indicator of smoking). The extent of intimal surface involved with both fatty streaks and raised lesions increased with age in all arterial segments of all sex and race groups. Women had a greater extent of fatty streaks in the abdominal aorta than men, but women and men had about an equal extent of raised lesions. Women and men had a comparable extent of fatty streaks in the right coronary artery, but women had about half the extent of raised lesions. Blacks had a greater extent of fatty streaks than whites, but blacks and whites had a similar extent of raised lesions. VLDL plus LDL cholesterol concentration was associated positively and HDL cholesterol was associated negatively with the extent of fatty streaks and raised lesions in the aorta and right coronary artery. Smoking was associated with more extensive fatty streaks and raised lesions in the abdominal aorta. All three risk factors affected atherosclerosis to about the same degree in both sexes and both races. Primary prevention of atherosclerosis by controlling these adult coronary heart disease risk factors is applicable to young men and women and to young blacks and whites.
Normolipidemic Subjects With Low HDL Cholesterol Levels Have Altered HDL Subpopulations
Authors: Asztalos Bela F. , Lefevre Michael , Foster Theda A. , Tulley Richard , Windhauser Marlene , Wong Laurence , Roheim Paul S.
Published: June 2011

Abstract
Epidemiological studies have established that plasma concentration of HDL is inversely correlated with the risk of coronary heart disease, even in the absence of increased LDL cholesterol levels. We postulate that specific HDL subpopulations may be responsible for antiatherogenic properties of HDL. HDL subpopulations were quantitated by two-dimensional gel electrophoresis in 79 normolipidemic healthy male subjects. To eliminate the influence of diet, volunteers consumed an average American diet for 6 weeks. After the diet period, subjects were stratified according to their HDL cholesterol (HDL-C) levels to low HDL-C <0.91 mmol/L (<35 mg/dL), medium >0.91<1.30 mmol/L (>35 <50 mg/dL), and high ≥1.30 mmol/L (≥50 mg/dL) groups. Plasma triglycerides and insulin levels were in the normal range, but subjects with low HDL-C levels had higher concentrations of plasma triglycerides and insulin than subjects with medium or high HDL-C concentrations. The absolute concentration (mg/dL) of apoA-I in the largest α-migrating HDL subpopulation (α
1
) was (
P
<.01) lower in the low HDL-C subjects compared with the medium and high HDL-C groups. The relative concentration (percent distribution) of apoA-I was decreased (
P
<.01) in α
1
and increased (
P
<.01) in α
3
subpopulations. A positive correlation between HDL-C and α
1
(
P
<.001) and a negative correlation between HDL-C and α
3
were observed. The inverse correlation of apoA-I distribution (relative concentration) between α
1
and α
3
suggests an interconversion of α
1
and α
3
subpopulations, possibly by cholesteryl ester transfer protein. Pre-β subpopulations showed an inverse trend with HDL-C, while the pre-α subpopulation behaved similarly to the α-migrating subpopulation. Colocalization of apoA-I and apoA-II particles in the different HDL subpopulations demonstrated that α
1
, pre-β
1
, and pre-β
2
subpopulations are apoA-I–only particles rather than apoA-I:A-II particles.
Factor VII Polymorphisms in Populations With Different Risks of Cardiovascular Disease
Authors: de Maat Moniek P.M. , Green Fiona , de Knijff Peter , Jespersen Jørgen , Kluft Cornelis
Published: June 2011

Abstract
Increased plasma factor VII coagulant activity (FVII:C) has been associated with the risk of ischemic heart disease (IHD). Differences in plasma FVII:C among individuals are associated with three common polymorphisms in the FVII gene. Therefore, we investigated FVII polymorphisms in four populations that differ in their risk of developing cardiovascular disease, namely, Europeans, Greenland Inuit, Gujarati Indians, and Afrocaribbeans. We studied (1) the promoter polymorphism, which is the result of a decanucleotide insertion in the FVII promoter at position −323 from the start of translation; (2) the hypervariable region 4 polymorphism (HVR4), which is the result of a variable number of tandem repeats in intron 7; and (3) the RQ353 polymorphism, a guanine-to-adenine substitution in the position of the codon for amino acid 353 resulting in an amino acid replacement of arginine (R) by glutamine (Q) in the FVII protein. The frequencies of these three polymorphisms and their linkage disequilibrium were different in the four populations studied. The frequencies of the alleles associated with higher plasma FVII:C were lower in the Europeans than in the Inuit, a population with a lower incidence of IHD. There was an association between both the promoter polymorphism and the RQ353 polymorphism and the plasma FVII:C in the Europeans, the Inuit, and the Gujarati Indians, and an association only between the RQ353 polymorphism and plasma FVII:C in the Afrocaribbeans. Only in the Inuit was the HVR4 polymorphism associated with plasma FVII:C. In multiple regression analysis, the additional information provided by the promoter polymorphism when the other polymorphisms were already included in the model was the most pronounced, suggesting that the promoter polymorphism may be the functional mutation having the greatest effect on determining plasma FVII:C.
Evaluation of Endothelial Shear Stress and 3D Geometry as Factors Determining the Development of Atherosclerosis and Remodeling in Human Coronary Arteries in Vivo
Authors: Krams R. , Wentzel J.J. , Oomen J.A.F. , Vinke R. , Schuurbiers J.C.H. , de Feyter P.J. , Serruys P.W. , Slager C.J.
Published: June 2011

Abstract
The predilection sites of atherosclerotic plaques implicate rheologic factors like shear stress underlying the genesis of atherosclerosis. Presently no technique is available that enables one to provide 3D shear stress data in human coronary arteries in vivo. In this study, we describe a novel technique that uses a recently developed 3D reconstruction technique to calculate shear stress on the endothelium with computational fluid dynamics. In addition, we calculated local wall thickness, the principal plane of curvature, and the location of plaque with reference to this plane, relating these results to shear stress in a human right coronary artery in vivo. Wall thickness and shear stress values for the entire vessel for three inflow-velocity values (10 cm/second, 20 cm/second, and 30 cm/second equivalents with the Reynolds numbers 114, 229, and 457) were as follows: 0.65±0.37 mm (n=1600) and 19.6±1.7 dyne/cm
2
; 46.1±8.1 dyne/cm
2
and 80.1±16.8 dyne/cm
2
(n=1600). Curvature was 25±9 (m
−1
), resulting in Dean numbers 20±8; 46±16, and 93±33. Selection of data at the inner curvature of the right coronary artery provided wall thickness values of 0.90±0.41 mm (n=100), and shear stress was 17±17, 38±44, and 77±54 dyne/cm
2
(n=100), whereas wall thickness values at the outer curve were 0.37±0.17 mm (n=100) and shear stress values were 22±17, 60±44, and 107±79 dyne/cm
2
(n=100). These findings could be reconciled by an inverse relationship between wall thickness and shear stress for each velocity level under study. For the first time for human vessels in vivo, evidence is presented that low shear stress promotes atherosclerosis. As the method is nondestructive, it allows repeated measurements in the same patient and will provide new insights in the progress of atherosclerosis.
Large Versus Small Unilamellar Vesicles Mediate Reverse Cholesterol Transport In Vivo Into Two Distinct Hepatic Metabolic Pools
Authors: Rodrigueza Wendi V. , Mazany Kirstin D. , Essenburg Arnold D. , Pape Michael E. , Rea Thomas J. , Bisgaier Charles L. , Williams Kevin Jon
Published: June 2011

Abstract
Phospholipid liposomes are synthetic mediators of “reverse” cholesterol transport from peripheral tissue to liver in vivo and can shrink atherosclerotic lesions in animals. Hepatic disposal of this cholesterol, however, has not been examined. We compared hepatic effects of large (≈120-nm) and small (≈35-nm) unilamellar vesicles (LUVs and SUVs), both of which mediate reverse cholesterol transport in vivo but were previously shown to be targeted to different cell types within the liver. On days 1, 3, and 5, rabbits were intravenously injected with 300 mg phosphatidylcholine (LUVs or SUVs) per kilogram body weight or with the equivalent volume of saline. After each injection, LUV- and SUV-injected animals showed large increases in plasma concentrations of unesterified cholesterol, indicating mobilization of tissue stores. After hepatic uptake of this cholesterol, however, SUV-treated animals developed persistently elevated plasma LDL concentrations, which by day 6 had increased to more than four times the values in saline-treated controls. In contrast, LUV-treated animals showed normal LDL levels. By RNase protection assay, SUVs suppressed hepatic LDL receptor mRNA at day 6 (to 61±4% of control, mean±SEM), whereas LUVs caused a statistically insignificant stimulation. Hepatic HMG-CoA reductase message was also significantly suppressed with SUV, but not LUV treatment, and hepatic 7α-hydroxylase message showed a similar trend. These data on hepatic mRNA levels indicate that SUVs, but not LUVs, substantially perturbed liver cholesterol homeostasis. We conclude that LUVs and SUVs mobilize peripheral tissue cholesterol and deliver it to the liver, but to distinct metabolic pools that exert different regulatory effects. The effects of one of these artificial particles, SUVs, suggest that reverse cholesterol transport may not always be benign. In contrast, LUVs may be a suitable therapeutic agent, because they mobilize peripheral cholesterol to the liver without suppressing hepatic LDL receptor mRNA and without provoking a subsequent rise in plasma LDL levels.